The Pituitary (Pituitary (Melmed))

The Pituitary

Notice: The indications and dosages of all drugs in this book have been recommended in the medical literature and conform to the practices of the general community. The medications described do not necessarily have specific approval by the Food and Drug Administration for use in

the diseases and dosages for which they are recommended. The package insert for each drug should be consulted for use and dosage as approved by the FDA. Because standards for usage change, it is advisable to keep abreast of revised recommendations, particularly those concerning new drugs.

The Pituitary EDITED BY

SHLOMO MELMED, Senior Vice-President, Academic Affairs Cedars-Sinai Medical Center Professor and Director, Burns & Allen Research Institute UCLA School of Medicine Los Angeles, California

SECOND EDITION

Blackwell Science

MD

© 2002 by Blackwell Science, Inc. Editorial Offices: Commerce Place, 350 Main Street, Malden, Massachusetts 02148, USA Osney Mead, Oxford OX2 0EL, England 25 John Street, London WC1N 2BS, England 23 Ainslie Place, Edinburgh EH3 6AJ, Scotland 54 University Street, Carlton, Victoria 3053, Australia Other Editorial Offices: Blackwell Wissenschafts-Verlag GmbH, Kurfürstendamm 57, 10707 Berlin, Germany Blackwell Science KK, MG Kodenmacho Building, 7-10 Kodenmacho Nihombashi, Chuo-ku, Tokyo 104, Japan Iowa State University Press, A Blackwell Science Company, 2121 S. State Avenue, Ames, Iowa 50014-8300, USA

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Contents

Contributors vii Preface

Section 3. PITUITARY TUMORS

x

10.

Pituitary Surgery 405 Rudolf Fahlbusch, Michael Buchfelder and Panos Nomikos

11.

Acromegaly 419 Shlomo Melmed

12.

Prolactinoma 455 Mark E. Molitch

13.

Cushing’s Disease 496 Xavier Bertagna, Marie-Charles Raux-Demay, Brigitte Guilhaume, François Girard and Jean-Pierre Luton

14.

Thyrotropin-secreting Pituitary Tumors 561 Yona Greenman and Shlomo Melmed

15.

Gonadotroph Adenomas Peter J. Snyder

16.

Nonpituitary Tumors of the Sellar Region 592 Steffen Albrecht, Juan M. Bilbao and Kalman Kovacs

Section 1. HYPOTHALAMIC–PITUITARY FUNCTION 1.

2.

Functional Anatomy of the Hypothalamic Pituitary Axis 3 Sylvia L. Asa, Kalman Kovacs and Shlomo Melmed Adrenocorticotropin 45 Mark A. Herman and Joseph A. Majzoub

3.

Growth Hormone 79 Vivien S. Herman-Bonert and Shlomo Melmed

4.

Prolactin 119 Mark E. Molitch

5.

Thyroid-stimulating Hormone 172 Virginia D. Sarapura, Mary H. Samuels and E. Chester Ridgway

6.

Follicle-stimulating Hormone and Luteinizing Hormone 216 Shalender Bhasin, Charles E. Fisher and Ronald S. Swerdloff

7.

The Posterior Pituitary Daniel G. Bichet

279

Section 2. HYPOTHALAMIC–PITUITARY DYSFUNCTION 8.

The Hypothalamus 317 Glenn D. Braunstein

9.

Anterior Pituitary Failure Charles F. Abboud

349

575

Section 4. PITUITARY DISEASE IN SYSTEMIC DISORDERS 17.

Pituitary Function in Systemic Disorders 613 Harold E. Carlson

18.

The Pituitary Gland in Pregnancy and the Puerperium 628 Harold E. Carlson

19.

Drugs and Pituitary Function Harold E. Carlson

642 v

vi

Contents

Section 5.

DIAGNOSTIC PROCEDURES

20.

Pituitary Imaging 663 Barry D. Pressman

21.

Neuro-ophthalmologic Evaluation of Pituitary Disorders 687 Anthony C. Arnold

22. Index

Evaluation of Normal Pituitary Function 709 Gillian L. Booth, Afshan Zahedi and Shereen Ezzat 727

Contributors

Charles F. Abboud, MD David Eisenberg Professor of Medicine Consultant, Internal Medicine and Endocrinolgy Mayo Clinic, Mayo Medical School Rochester, MN Steffen Albrecht, MD FRCP(C) Department of Pathology Sir Mortimer B. Davis Jewish General Hospital Montreal, Canada Anthony C. Arnold, MD Associate Professor of Ophthalmology and Chief, Neuro-Ophthalmology Division Jules Stein Eye Institute, UCLA Department of Ophthalmology Los Angles, CA Sylvia L. Asa, MD, PhD Associate Professor of Pathology Mount Sinai Hospital University of Toronto Toronto, Canada Xavier Bertagna, MD Professor of Endocrinology Clinique des Maladies Endocriniennes et Metaboliques Hôpital Cochin Paris, France Shalender Bhasin, MD Chief, Division of Endocrinology, Metabolism, and Molecular Medicine Professor of Medicine Charles R. Drew University School of Medicine and Science Los Angeles, CA

Daniel G. Bichet, MD, FRCP(C) Service de néphrologie et Centre de recherche Hôpital du Sarcé-Coeur de Montréal Department of Medicine Université de Montréal Montréal, Quebec Canada Juan M. Bilbao, MD, FRCP(C) Neuropathologist, Department of Pathology Saint Michael’s Hospital University of Toronto Toronto, Canada Gillian L. Booth, MD Department of Medicine University of Toronto Mount Sinai Hospital Toronto, Canada Glenn D. Braunstein, MD Chairman, Department of Medicine Cedars-Sinai Medical Center Professor of Medicine UCLA School of Medicine Los Angles, CA Michael Buchfelder, MD Associate Professor Department of Neurosurgery University of Erlangen-Nürnberg Erlangen, Germany vii

viii

Contributors

Harold E. Carlson, MD Chief, Endocrinology Section Northport Veterans Affairs Hospital, Northport Professor of Medicine State University of New York at Stony Brook Stony Brook, NY

Kalman Kovacs, MD, PhD, DSc, FRCP(C), FCAP, FRC Path. Professor of Pathology Saint Michael’s Hospital University of Toronto Toronto, Canada

Shereen Ezzat, MD, FRCP(C), FACP Assistant Professor of Medicine University of Toronto Consultant Endocrinologist Wellesley Hospital Toronto, Canada

Jean-Pierre Luton, MD Professor of Endocrinology and Director Clinique des Maladies Endocriniennes et Metaboliques Hôpital Cochin Dean, Cochin Medical School University René Descartes Paris, France

Rudolf Fahlbusch, MD Professor of Neurosurgery Department of Neurosurgery University of Erlangen-Nürnberg Erlangen, Germany Charles Ellis Fisher, MD Division of Endocrinology and Metabolism King Drew Medical Center Los Angeles, CA François Girard, MD Professor of Physiology and Director Laboratoire d’Explorations Fonctionnelles Endocriniennes Hôpital Trousseau Paris, France Yona Greenman, MD Tel-Aviv Sourasky Medical Center Tel-Aviv, Israel Brigitte Guilhaume, MD Clinique des Maladies Endocriniennes et Metaboliques Hôpital Cochin Paris, France Mark A. Herman, MD Fellow in Medicine Department of Internal Medicine University of Texas Southwestern Medical Center Dallas, Texas Vivien S. Herman-Bonert, MD Division of Endocrinology Cedars-Sinai Medical Center Associate Professor of Medicine UCLA School of Medicine Los Angeles, CA

Joseph A. Majzoub, MD Chief, Division of Endocrinology Department of Pediatrics Children’s Hospital Professor of Pediatrics Harvard Medical School Boston, MA Shlomo Melmed, MD Senior Vice-President, Academic Affairs Director, Burns & Allen Research Institute Associate Dean, UCLA School of Medicine Cedars-Sinai Medical Center Los Angeles, CA Mark E. Molitch, MD Professor of Medicine Center for Endocrinology, Metabolism, and Molecular Medicine Northwestern University Medical School Chicago, IL Panos Nomikos, MD Neurosurgical Fellow Department of Neurosurgery University of Erlangen-Nürnberg Erlangen, Germany Barry D. Pressman, MD, FACR Chief, Section of Neuroradiology and Chairman, Department of Imaging Cedars-Sinai Medical Center Los Angles, CA Marie-Charles Raux-Demay, MD Laboratoire d’Explorations Fonctionnelles Endocriniennes Hôpital Trousseau Paris, France

Contributors

Chester E. Ridgway, MD Professor of Medicine and Head, Division of Endocrinology University of Colorado Health Sciences Center Denver, CO Mary H. Samuels, MD Assistant Professor of Medicine Division of Endocrinology Oregon Health Science University Portland, OR Virginia D. Sarapura, MD Assistant Professor of Medicine Department of Medicine, Division of Endocrinology University of Colorado Health Sciences Center Denver, CO

Peter J. Snyder, MD Professor of Medicine University of Pennsylvania School of Medicine Philadelphia, PA Ronald S. Swerdloff, MD Professor of Medicine and Chief, Division of Endocrinology Department of Medicine Harbor/UCLA Medical Center UCLA School of Medicine Torrance, CA Afshan Zahedi, MD Department of Medicine University of Toronto Mount Sinai Hospital Toronto, Canada

ix

Preface

The second edition of The Pituitary follows the successful initial 1995 debut of this comprehensive text devoted to understanding pathogenesis and treatment of pituitary disorders. The new edition is extensively revised to reflect the wealth of novel information derived from advances in molecular biology, biochemistry and therapeutics as they apply to the pituitary gland. Notably, a new chapter devoted to pituitary surgery has been added to complement a comprehensive overview of management options for patients harboring pituitary tumors. The wide spectrum of clinical disorders emanating from disordered function of the “master gland” are described in detail by experts in the field. Furthermore, fundamental mechanisms underlying disease pathogenesis are presented to provide the reader with an in-depth understanding of mechanisms underlying pituitary hormone secretion and action.

Thus the volume continues to reflect the cogent blend of basic science and clinical medicine which was the successful hallmark of the first edition. I am especially indebted to my erudite colleagues for their scholarly contributions and dedicated efforts in compiling this body of knowledge for physicians and scientists dedicated to caring for patients with pituitary disorders. Our desire is to provide medical students, clinical and basic endocrinology trainees, endocrinologists, internists pediatricians, gynecologists and neurosurgeons with a comprehensive single text devoted to the science and art of pituitary medicine. Shlomo Melmed, MD Los Angeles 2002

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Hypothalamic–Pituitary Function

C h a p t e r

1 Functional Anatomy of the Hypothalamic Pituitary Axis Sylvia L. Asa Kalman Kovacs Shlomo Melmed

INTRODUCTION

Historical Review Descartes was the first to recognize that the brain was an organ integrating the functions of the mind and body [1]. The association was reinforced by Zander [2] who noted the connection between the adrenals and the brain, referring to observations on the absence of the adrenal cortex in anencephaly as recorded by Morgagni in 1733, Soemmering in 1792, and Meckel in 1802. It was only in 1849 that direct evidence of a role for the hypothalamus in endocrinology was provided by Claude Bernard [3] when he demonstrated that injury to the floor of the fourth ventricle, the “piqûre diabetique,’ caused polyuria and glucosuria; subsequently, numerous studies in the late 19th century and early 20th century confirmed that the hypothalamic–posterior pituitary system was the site of production of a major osmoregulatory substance. Galen (129–201 ad) had described the pituitary as the site of drainage of phlegm from the brain to the nasopharynx. Soon after the description of acromegaly by Pierre Marie in 1886 [4], the association of acromegaly with pituitary tumor was noted by Minkowski in 1887 [5], and the recognition of endocrine functions of the pituitary followed rapidly thereafter, with major contributions by Cushing [6] and Simmonds [7]. The early part of the 20th century saw the identification, isolation and characterization of the hormones of the anterior pituitary. Their regulation by the hypothalamus was the subject of a landmark monograph by Harris in 1948 [8]. The concept that neurons could release secretory materials into the bloodstream, proposed by Du Bois Reymond

in 1877 [9] and Schiefferdecker in 1905 [10], was revolutionary. The anatomic basis of a hypothalamic–pituitary vascular connection was already known to exist in the form of a system of capillaries, which were identified joining the medial basal hypothalamus to the anterior pituitary by Lieutaud in 1742 [11] and von Luschka in 1860 [12] and given the name “portal plexus” by Popa and Fielding in 1930 [13]. These vessels were thought at first to carry blood from the pituitary upwards to the hypothalamus; however, subsequent studies confirmed that they provided the main blood supply to the adenohypophysis. The concept of neurosecretion was suggested in the 1940s by Scharrer and Scharrer [14] and Bargmann and Scharrer [15], who demonstrated that peptide synthesized by neurons in the supraoptic and paraventricular nuclei of the hypothalamus passed through nerve fibers, was stored in nerve endings in the posterior pituitary and subsequently was released into the general circulation; the analysis and synthesis of oxytocin and vasopressin in 1954 [16] led to a Nobel Prize for du Vigneaud. In the 1950s and 1960s, evidence for the presence of hypothalamic releasing and inhibiting factors accumulated [17]. The isolation and characterization of the first of these yielded shared Nobel prizes for Guillemin and Schally in 1977 and by the mid 1980s, the isolation and characterization of many had been achieved [18–26].

Methodology Progress in the understanding of hypothalamic and pituitary endocrinology has utilized numerous techniques which have allowed the identification of hormones, their isolation and characterization, their localization, and recognition of their cell of origin. At the beginning of the 20th century, Benda [27], Erdheim [28], Cushing [6], and others [29,30] utilized histologic staining methods for the investigation of the human pituitary. Morphologic studies of the hypothalamus were 3

4

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Hypothalamic–Pituitary Function

dependent on conventional neurohistologic methods to demonstrate neurosecretory material and the silver stains that allowed visualization of neuronal cell processes [31]. The tinctorial properties of cell cytoplasm using hematoxylin–eosin, hematoxylin–phloxine, or hematoxylin– phloxine–saffron methods allowed the distinction of three cell types in the adenohypophysis. Clinical observations, correlated with morphologic changes, led to the classification of acidophils as cells thought to produce growth hormone (GH), basophils as those believed to contain adrenocorticotropin (ACTH) and chromophobes as functionally inactive cells. In the 1930s, Rasmussen [32] quantitated the three cell types in relation to sex, age, and body weight and determined their distributions within the gland. In 1940, Romeis [33] used a modification of the trichrome technique to establish that more than three cell types were identifiable in the adenohypo-physis. Application of the periodic acid–Schiff (PAS) method distinguished the PASpositive corticotrophs, thyrotrophs and gonadotrophs; similar results were obtained with lead hema-toxylin. Various trichrome stains, the aldehyde fuchsin and aldehyde thionin techniques were used to separate basophils [34] and the addition of Herlant’s erythrosin [35] and Brookes’ carmoisine [36] were useful to distinguish lactotrophs from the orange G-positive somatotrophs. Physiologic and biochemical studies provided evidence that six different hormones are produced by the anterior pituitary; however, histologic techniques failed to correlate cell type with clinical hormone production or secretory function. The search for correlations between structure and function led to the application of novel techniques including immunocytology [37,38] and electron microscopy [39,40] to the study of pituitary morphology. Ultrastructural immunocytochemistry [41], greatly improved by the use of the immunogold technique [40,42], and the application of the reverse hemolytic plaque assay [43], tissue culture methods [44] and molecular probes [45], have made pituitary and hypothalamic morphology significant for assessing pituitary function and pathology. HYPOTHALAMUS AND POSTERIOR PITUITARY

Topographic Anatomy The hypothalamus is a phylogenetically primitive structure. Understanding of its role in endocrine regulation is largely derived from animal studies; early data regarding functional localization were based on selective stimulation and ablation experiments. However, there are considerable anatomic and physiologic species variations which make extrapolation to the human difficult. Only the advent of sophisticated neurophysiologic methods and the recent applications of immunocytochemistry, as well as experiments of nature in which careful analyses of the location of pathologic lesions are performed, have allowed structure–function correlations in the human hypothalamus.

The hypothalamus is a poorly defined anatomic region which constitutes less than 1% of brain volume and weighs approximately 5 g [46,47]. The superior border of this region is the hypothalamic sulcus which courses from the interventricular foramen to the cerebral aqueduct, demarcating the hypothalamus from the thalamus (Fig. 1.1). The anterior delineation is roughly defined as a line through the anterior commissure, lamina terminalis, and optic chiasm. Posteriorly, it is bordered on the superior aspect by the midbrain tegmentum and inferiorly by the mamillary bodies. The lateral borders are defined conventionally as the substantia innominata, the internal capsule, the subthalamic nucleus, and the cerebral peduncle. The inferior aspect of the hypothalamus, known as the tuber cinereum, contains the median eminence and gives rise to the infundibulum, the neural stalk and the posterior lobe of the pituitary.

Vascular Supply A significant number of afferent and efferent signals of the hypothalamus are not neural, but rather represent bloodborne information, such as temperature, osmotic pressure, hormones, and glucose levels, thus endorsing the importance of the vascular supply of this area. The blood supply of the hypothalamus is derived from small arterial branches of the circle of Willis and the superior hypophysial artery. It is divided into three parts: (i) an anterior group which arises from the internal carotid artery, the anterior cerebral artery, and the anterior portion of the posterior communicating artery; (ii) an intermediate group from the posterior communicating artery; and (iii) a posterior group from the posterior communicating artery, posterior cerebral, and basilar arteries. Individual hypothalamic nuclei receive blood from more than one small artery. The microvascular pattern of the hypothalamus resembles that of the remainder of the brain with the exception of the magnocellular nuclei, the supraoptic and paraventricular nuclei. These are some of the most richly vascularized structures in the brain, in which the neurons are intimately associated with capillaries. The capillary endothelium is fenestrated and the blood–brain barrier is absent in many areas of the hypothalamus, including the subfornical organ, the organum vasculosum of the lamina terminalis (OVLT), the median eminence, and the neurohypophysis. The arterial supply of the median eminence and posterior pituitary has been extensively studied [48–51] (Fig. 1.2). It is derived from two (or three, in some species) paired arteries that arise from the intracranial portions of the internal carotid arteries. The superior hypophysial arteries branch into a plexus of small arteries that surrounds the upper half of the stalk (the external plexus), giving rise to a mesh of capillaries, and the gomitoli (the internal plexus). The latter are unique vascular structures composed of a central muscular artery surrounded by a spiral of capillaries, the arteriole communicating with the capillaries by way of small

Chapter 1

Functional Anatomy of the Hypothalamic Pituitary Axis

5

FIGURE 1.1. Schematic representation of the human hypothalamus and pituitary, the disposition of hypothalamic nuclei and principal fiber tracts. From Scheithauer [47]

orifices surrounded by muscular sphincters. The gomitoli, which measure 1–2 mm in length and 0.1 mm in width, are found in large numbers in the infundibulum and proximal hypophysial stalk. Flow through these complex structures continues on through the portal vessels to the adenohypophysial capillaries. Although their function is not certain, the unique structure of gomitoli suggests that they may affect the rate of blood flow to the anterior pituitary, thereby influencing the entry of hypothalamic regulatory hormones into the portal circulation. In some individuals, a pair of middle hypophysial arteries also contributes to the trabecular or loral arteries which descend from the superior hypophysial arteries along the external surface of the pituitary stalk. These then divide to contribute to the subcapsular artery and the artery of the fibrous core (which provide a minor contribution to the blood supply of the adenohypophysis) and return upwards along the pituitary stalk as the long stalk arteries. The inferior hypophysial

arteries enter the sella just beneath its diaphragm and supply the pituitary capsule, the neural lobe, and the lower pituitary stalk. They enter the intralobar groove where they divide into ascending and descending branches that join their opposite partners to form an arterial circle about the neural lobe. They also give off a branch to the lower pituitary stalk, the communicating artery, which anastomoses with the trabecular arteries. Notably, the capillaries of the neurohypophysis are fenestrated and lie outside the blood–brain barrier. The earliest studies of the hypophysial portal vasculature assumed that blood flowed from the pituitary upwards to the hypothalamus [13]. Subsequently, it was found that the predominant blood flow is from the hypothalamus to the adenohypophysis, carrying hypothalamic regulatory factors. Recent studies suggest that the direction of blood flow is variable and there may be a component of reverse flow within the neurohypophysis, providing a route by which

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FIGURE 1.2. Schematic representation of the blood supply of the hypothalamus and pituitary. From Scheithauer [47]

neuro- and adenohypophysial hormones gain access to the long portal system, the pars tuberalis of the adenohypophysis, the median eminence, and the cerebrospinal fluid of the third ventricle [50,51].

Nerve Supply A number of afferent neural pathways provide the hypothalamus with input from the forebrain, limbic system, visual cortex, thalamus, and brain stem [47,52]. The median forebrain bundle, a large unmyelinated structure lateral to the fornix, carries information to and from the brain stem and provides important input from the limbic system and olfactory forebrain structures. Olfactory connections are more extensively from the piriform cortex and

amygdaloid nuclear complex via the stria terminalis and ventral amygdalofugal pathway. The hippocampal–hypothalamic tract originates in the hippocampus, a limbic structure, and enters the anterolateral portion of the mamillary bodies. The amygdalo–hypothalamic tract provides entry of emotional data from the amygdaloid nucleus into the hypothalamus. The retino–hypothalamic tract originates in the ganglion cells of the retina and affects the pineal gland, playing a role in the regulation of circadian rhythms. In addition, a small number of cortico–hypothalamic fibers from the posterior orbital gyrus of the frontal lobe is a potential source of input from the neocortex into the hypothalamus.

Chapter 1

The hypothalamus sends efferent projections to the forebrain, brain stem, spinal cord and neurohypophysis. The median forebrain bundle carries fibers to the septal nuclei, integrating visceral and olfactory functions. The dorsal longitudinal fasciculus carries efferents to the midbrain tegmentum where it innervates visceral and sympathetic motor neurons. Fibers emanating from the mamillary body divide into the mamillo–thalamic tract, serving the anterior nucleus of the thalamus and memory function, and the mamillotegmental tract which projects to the midline tegmentum. Efferents from the ventral medial nucleus project to the amygdaloid nucleus via the stria terminalis. The hypothalamo–hypophysial tract consists primarily of nerve fibers from the supraoptic and paraventricular nuclei carrying vasopressin, oxytocin and their neurophysins to the posterior lobe of the pituitary, where the hormones are released into capillaries. The tubero–infundibular tract, originating from neurosecretory neurons which produce hypophysiotropic hormones, projects from several nuclei to the median eminence where the hormones are released into the hypophysial portal vascular system.

Nuclei and Nucleoinfundibular Pathways Within the hypothalamus, distinct clusters of neurons are called “nuclei.” The hypothalamic nuclei are generally divided into four anatomic regions, the preoptic, supraoptic and lateral, tuberal, and mamillary regions. In general, the nuclei are topographically discrete in many species and, although relatively well demarcated in the human fetus, they are poorly defined in the mature human hypothalamus [46]. In a few cases, the existence of some nuclei is inferred entirely from studies of experimental animals. A schematic illustration is provided in Fig. 1.1. Structure–function correlations are difficult because of cellular heterogeneity of many hypothalamic nuclei. Any given hypothalamic hormone is often produced in more than one nucleus and, in many cases, a single nucleus may express more than one hormone. The physiologic roles of many nuclei remain unknown. The most anterior nuclei are the paired medial and lateral nuclei that have been associated with autonomic function, particularly temperature control and olfaction. The suprachiasmatic nucleus is found in the preoptic area dorsal to the optic chiasm and anterior to the supraoptic nucleus. This sexually dimorphic cell group shows a striking decrease in volume and cell number with age [53]. It is located in an area that is essential for gonadotropin release and sexual behavior in lower animals and may play a role in maintaining circadian rhythms. It is thought that the nucleus also plays a role in the sexual differentiation of the brain which, in the absence of male gonadal hormones, remains female but, if exposed to these at a critical stage in development, becomes male [54]. It is also associated with fibers of the supraoptic commissure, and receives afferents from the retina and the lateral geniculate bodies.

Functional Anatomy of the Hypothalamic Pituitary Axis

7

The anterior hypothalamic nucleus is composed of small neurons which mediate parasympathetic effects; it has connections to the insular cortex, substantia innominata and thalamus. The lateral hypothalamic nuclei are poorly defined areas composed of larger neurons which receive fibers from and contribute efferents to the median forebrain bundle. The paraventricular nuclei lie adjacent to the third ventricle and ventromedial to the fornix. They are composed mainly of large “magnocellular” neurons along with the supraoptic nuclei and also contain a number of “parvicellular” neurons. Efferent fibers to the pituitary stalk terminate in the posterior lobe of the pituitary. These nuclei are a major site of oxytocin and vasopressin synthesis. In patients with traumatic or surgical stalk section and in those with long-standing hypopituitarism, there is a marked reduction in the number of magnocellular neurons and stalk nerve fibers [46]; the parvicellular component remains. The supraoptic nuclei are the other paired magnocellular nuclei of the hypothalamus; they have no significant parvicellular component. They overlie the anterior optic tract and posteriorly are divided by the optic tract into anterolateral and posteromedial components, united by a thin stream of cells. Patients with stalk section or long-standing hypopituitarism also have atrophy of these nuclei [55]. The dorsomedial and ventromedial nuclei are involved in autonomic function and emotional behavior. They are situated between the tuber cinereum and paraventricular nuclei. Stimulation of the dorsomedial and destruction of the ventromedial nuclei produces rage in experimental animals. The ventromedial nuclei are concerned with control of balance between hunger and satiety. Destruction of the ventromedial nucleus results in obesity [56,57]; conversely, destruction of the ventrolateral nucleus, known as the “feeding center,” is associated with anorexia and cachexia [56]. These nuclei show a large number of afferent connections from olfactory and retinal fibers, the reticular formation, and the nucleus of the solitary tract which receives input from the vagus. Afferents from the cortex enter by way of the thalamus. Ventral to the third ventricle and paraventricular nuclei lies the arcuate (infundibular) nucleus which is another important component of the hypophysiotropic region and plays a major role in the modulation of anterior pituitary function. The subventricular nucleus lies on the floor of the third ventricle posterior to the arcuate and anteromedial to the tuberal nuclei. This usually parvicellular nucleus shows marked hypertrophy of the neurons which become magnocellular in postmenopausal women [58,59], in young women suffering from postpartum hypopituitarism with complete gonadal atrophy [60], in hypogonadal men and women, in starvation [61], after hypophysectomy [62], and in late pregnancy. The neurons develop a distinctive nucleolar change considered a manifestation of feedback effect; a similar change is also observed in neurons of the arcuate

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Hypothalamic–Pituitary Function

nucleus [60]. In some of these conditions, the change is thought to reflect lack of estrogens [59,63,64]. The tuberal nuclei consist of irregularly grouped masses of large neurons inferior to the lateral nuclei that give rise to efferent fibers of the hypothalamus. After stalk section, a slight increase in coarsely granular basophilic cytoplasmic material may occur [46]. The posterior hypothalamic nucleus, situated between the third ventricle and the mamillo–thalamic tract, lies superior to the mamillary bodies. When stimulated, it produces sympathetic effects; it may play a role in temperature regulation and its large neurons may be the source of hypothalamic efferents that descend to the reticular formation of the brain stem. The paired mamillary nuclei and a variety of minor nuclei in the supramamillary area, including the nucleus intracalatus, form the most posterior portion of the hypothalamus. These nuclei integrate incoming information from the limbic system and the midbrain tegmentum and send out efferent fibers to the anterior nucleus of the thalamus and to the brain stem.

Embryology The forebrain of the human embryo can be identified at 4 weeks postconception; by 5 weeks it has divided into a central diencephalon and two lateral telencephalic structures. Three swellings appear in the diencephalon which represent the anlages of epithalamus, thalamus, and hypothalamus. The nuclei of the hypothalamus and the supraoptico-hypophysial tract are recognizable at 8 weeks of gestation [65]. The nerve cells originate from proliferating neuroblasts and form several closely associated nuclei in the hypothalamus. At 6 weeks, a thickening of neuroblasts projects caudally to become the median eminence and infundibulum and establishes direct contact with the upward invagination of the roof of the primitive oral cavity, Rathke’s pouch. By 3 months, the rudimentary neural lobe consists of a short wide sac with a thick wall and a lumen lined by columnar ependymal cells [66]. Proliferation of spongioblasts gives rise to the modified neuroglia of the neurohypophysis known as pituicytes while the ependymal cells become dispersed in the neuroglial substance. Downgrowth of unmyelinated axons of neurons of magnocellular nuclei of the hypothalamus reaches the median eminence by 3 months, passes through the stalk and terminates in the neural lobe by 6 months of gestation. Monoamine fluoresence can be detected at 10 weeks in hypothalamic nuclei and at 13 weeks in the median eminence [67]. The supraoptic nucleus matures earlier than the paraventricular nucleus; vasopressin is demonstrated earlier and in higher concentrations than oxytocin in the neural lobe [68]. Arginine vasopressin is identified by radioimmunoassay at 12 weeks of gestation [69]. Intracellular granules have been demonstrated in hypothalamic nuclei at 19 weeks and in the posterior lobe at 23 weeks of gestation. The

neurosecretory material is clearly visible in the supraoptic and paraventricular nuclei as well as the posterior lobe at 23–28 weeks of gestation [70–72]. Ultrastructurally, clusters of small clear vesicles and axon profiles have been identified in a fetus at 7.5 weeks of age. Granular vesicles become apparent in the axon profiles at 8.5 weeks of gestation. Fenestration of capillaries and expansion of perivascular spaces are noted with the appearance of intraaxonal vesicles [73]. Growth hormone-releasing hormone (GHRH) has been identified by immunohistochemistry in the fetal hypothalamus at 18 weeks of gestation [74]. Somatostatin (somatotropin release-inhibiting hormone; SRIH) is identified in fetal hypothalami in a large molecular form at 10 weeks of gestation and there is a gradual increase in both the number of somatostatin-containing neurons and the 14-amino acid form of this peptide with fetal age [75,76]. Dopamine is present in high concentrations in the fetal hypothalamus by 15 weeks of gestation [67]. Corticotropin-releasing hormone (CRH) has been detected in fetal median eminence at 16 weeks of gestation [77]. Thyrotropin-releasing hormone (TRH) and gonadotropin-releasing hormone (GnRH) are the first adenohypophysiotropic substances, being detectable in fetal hypothalami at 4.5 weeks and present in significant amounts by 10 weeks of gestation [78,79]. GnRH is localized by immunocytochemistry at 13 weeks and GnRH-containing nerve terminals are detected on portal vessels at 16 weeks [80]. Sexual dimorphism of GnRH concentration has been documented and correlates with sexual dimorphism of fetal gonadotropic hormones [81].

Functional Anatomy and Control of Anterior Pituitary Function Control of anterior pituitary hormone secretion is under a complex set of central and peripheral neural and chemical signals. The hypothalamus synthesizes and secretes unique releasing and inhibitory hormones that control the anterior pituitary hormones (Fig. 1.3). The polypeptide hormones are secreted into the hypothalamic pituitary portal vasculature, impinge upon specialized anterior pituitary cells and bind to specific surface receptors. The cell signal elicits regulation of pituitary trophic hormone gene transcription, translation, and/or secretion. The resultant secretion of anterior pituitary hormones into the systemic circulation depends upon a carefully controlled dynamic balance of hypothalamic hormone input, feedback regulation from peripheral target hormones, nonhormonal neurotransmitter agents, and paracrine or autocrine pituitary growth factors and peptides. These factors all contribute to the amount of each pituitary hormone secreted, as well as to their pattern of secretion. Complex feedback regulation loops interact to regulate the hypothalamic–pituitary axis. The target gland peripheral hormone usually exerts negative feedback regulation directly at the level of pituitary synthesis or secretion of the respective trophic hormone (Fig. 1.4). Pituitary

Chapter 1

Functional Anatomy of the Hypothalamic Pituitary Axis

9

FIGURE 1.3. Amino acid sequences of human hypothalamic hormones. CRH, corticotropin-releasing hormone; GHRH, growth hormone-releasing hormone; GnRH, gonadotropin-releasing hormone; SRIH, somatostatin; TRH, thyrotropin-releasing hormone.

FIGURE 1.4. General control of anterior pituitary hormone release by central and hypothalamic inputs and feedback regulation by peripheral and trophic hormones. ACTH, adrenocorticotropic hormone; CRH, corticotropin-releasing hormone; DA, dopamine; FSH, folliclestimulating hormone; GHRH, growth hormone-releasing hormone; GnRH, gonadotropin-releasing hormone; IGF-I, insulin-like growth factor-I; LH, luteinizing hormone; PRL, prolactin; SRIH, somatostatin; TRH, thyrotropin-releasing hormone; TSH, thyrotropin; VIP, vasoactive intestinal polypeptide.

trophic hormones may feed back in a very short loop to autoregulate their own secretion. They may also participate in short loop feedback regulation of the secretion of their respective hypothalamic releasing or inhibiting hormone. Finally, peripheral hormones may participate indirectly by feedback regulation of hypothalamic releasing and inhibiting hormones.

The central nervous system also influences the control of hypothalamic hormonal secretion by efferent signals emanating from exteroceptive organs including the ears, eyes, and skin, and from interoceptive senses of indices of metabolic and electrolyte homeostasis. The transduction of neuronal inputs to neurochemical signaling by the hypothalamus appears to occur in an oscil-

10

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latory fashion. The temporal nature of pituitary hormone secretion is a manifestation of this rhythmicity resulting in at least two patterns of circadian rhythm including sleeprelated hormone secretory cycles and 24-hour rhythm cycles entrained by a light–dark cycle. The peptidergic neurons of the hypothalamus are not classified into distinct cell types and there is no known correlation between cell structure and function. In fact, neurons of any anatomic area often contain more than one neuropeptide or candidate neurotransmitter. The functional anatomy of the hypothalamus is, therefore, best approached by discussion of each hormone or peptide. GHRH

The presence of the factor responsible for stimulating release of growth hormone (GH) was known for many years, but it was only in 1982 that GHRH was isolated and characterized [25,26]. It is now known to have two forms, a 40- and a 44-amino acid peptide; the active portion of the molecule is the 29-amino acid N-terminus. GHRH binds to the surface of the somatotrophs. The intracellular actions of GHRH are probably mediated by cyclic adenosine monophosphate (cAMP), resulting in stimulation of GH gene transcription, translation, and secretion. Somatostatin attenuates the secretion of GH by the somatotroph. GH stimulates predominantly the liver to produce insulin-like growth factor-I (IGF-I) which acts as a target hormone to induce tissue growth. GH itself also acts directly on the chondrocytes and possesses antiinsulin metabolic activity. Immunocytochemical studies have identified GHRH in neuronal cell bodies of the arcuate nucleus [82,83] (Fig. 1.5). Nerve fibers from this nucleus project mainly to the median eminence, are found in the external zone, and terminate on capillaries of the plexus of the hypothalamohypophysial portal system. In monkey hypothalamus, small groups of immunoreactive neurons are also found in ventral

portions of the ventromedial nucleus. In the rat, scattered cells are also seen in the lateral basal hypothalamus, the medial and lateral portion of the ventromedial nucleus, and the dorsal medial and paraventricular nuclei [84,85]. The primary anatomic localization of GHRH to the arcuate nucleus is in agreement with earlier physiologic studies which indicated that a region including the ventromedial and arcuate nuclei was facilitatory in GH regulation. Extrapituitary effects of GHRH have been reported in the rat. Administration of small doses of the peptide into the cerebral ventricles induces behavioral sedation as well as an increase in food intake [86]. No extra-hypothalamic sites of GHRH synthesis are known to exist in the human under normal circumstances, but endocrine tumors of lung, pancreas, gut, adrenal medulla, and C-cells of thyroid can give rise to GHRH excess and acromegaly [87] (see Chapter 11). Somatostatin (SRIH)

A hypothalamic factor with GH release-inhibiting activity was first described in 1968 [88]. In 1973, Brazeau and colleagues [23] isolated and characterized a 14-amino acid peptide which they called somatostatin, or somatotropin release-inhibiting hormone (SRIH). This peptide was also shown to inhibit thyrotropin (thyroid-stimulating hormone; TSH) secretion and to produce a variety of other effects not related to its action on the pituitary. The hypothalamic distribution of SRIH shows marked species variation [89–92]. In the human hypothalamus, SRIH-containing neurons are concentrated in the anterior portion of the arcuate nucleus [90]. No positivity has been found in the paraventricular nucleus, unlike the distribution observed in rodents where SRIH is found in the paraventricular nucleus in a narrow field extending 1–2 mm from the midline. Nerve fibers staining for SRIH are observed in the medial basal hypothalamus; nerve endings are concentrated in the external zone of the median eminence and the neurovascular zone of the pituitary stalk. Fibers containing SRIH are also localized in the ventromedial, arcuate, suprachiasmatic, and paraventricular nuclei. Somatostatincontaining neurons store the hormone in dense core cytoplasmic vesicles which measure 80–110 nm in diameter and are found in both the cell bodies and the nerve endings [92]. Somatostatin is also found in extrahypothalamic regions of the rat brain, including the preoptic region, central gray mesencephalon, amygdala, olfactory area, and spinal cord. In humans it is detectable in cortex, brain stem, pineal, retina, optic nerve, auditory nerve, and spinal cord. Somatostatin is also found in pancreatic islet cells, gut endocrine cells, thyroid C-cells, salivary gland, and placenta [93–95]. Prolactin-inhibiting Factor

FIGURE 1.5. Neurons containing immunoreactive growth hormone-releasing hormone are localized by immunocytochemistry in the arcuate nucleus. (Magnification ¥64)

Prolactin (PRL) secretion by the adenohypophysis is known to be under tonic inhibition by the hypothalamus [96]. The PRL-inhibiting activity of dopamine is well documented and, currently, it is widely accepted that dopaminergic

Chapter 1

control is the main PRL regulatory function of the hypothalamus. Dopamine is the only nonpeptidergic hypothalamic compound with a well-defined hypophysiotropic function. It exerts primarily inhibitory effects on the lactotroph and inhibits both PRL gene transcription and lactotroph mitotic activity. PRL acts to induce lactation in concert with other peripheral hormones. Although TRH also stimulates lactotroph function, its effect is probably not physiologic. Vasoactive intestinal peptide (VIP) has also been shown to stimulate the production of PRL although its physiologic role has not yet been clarified. Dopamine has been localized to the arcuate and periventricular nuclei of the mediobasal hypothalamus as well as being widespread throughout the brain [97,98]. The medium-sized dopaminergic neurons are scattered among other neurosecretory cells and are further subdivided into the tuberoinfundibular group, with terminals in the median eminence and pituitary stalk, and the tuberohypophysial group, with ends terminating in the neural and intermediate lobes of the pituitary. The dopamine projections to the intermediate and neural lobes of the pituitary originate primarily in the anterior and central portions of the arcuate nucleus, respectively. Dopamine-containing axon terminals are abundant in the zona externa of the median eminence and are characterized by dense core vesicles that measure 15–120 nm in diameter. They do not appear to form true synaptic connections. CRH

Despite being the first hypothalamic regulatory factor postulated, CRH was among the last identified. In 1981, a 41-amino acid peptide was extracted from the ovine hypothalamus, characterized and shown to stimulate specifically the release of ACTH and other pituitary peptides derived from proopiomelanocortin (POMC) [24]. CRH binds to the surface of the corticotroph and elicits transcription of the POMC gene, whose products include ACTH and bendorphin. ACTH stimulates the adrenal gland to synthesize corticosteroids which are critical for essential stress responses and cellular homeostasis. Antibodies against CRH have localized this peptide to neuronal cell bodies, mainly in the parvocellular portion of the paraventricular nucleus but also in a few magnocellular neurons [99–104]. In the rat hypothalamus, scattered cell bodies containing CRH are also detected in the lateral preoptic and hypothalamic areas and in some portions of the dorsal medial nucleus. CRH-immunoreactive fibers are observed in the area of the paraventricular nucleus, the neurovascular zone of the median eminence, the pituitary stalk, and the neurohypophysis. CRH is colocalized with arginine vasopressin in a subpopulation of magnocellular neurons of the paraventricular nucleus [103,104]; it is found together with enkephalin, peptide histidine isoleucine (PHI, a member of the secretin–glucagon family) and neurotensin in parvocellular neurons as well as in a discrete subset of oxytocinergic magnocelluar neurons [104]. Adrenalectomy

Functional Anatomy of the Hypothalamic Pituitary Axis

11

increases CRH immunoreactivity in parvocellular neurons of the paraventricular nucleus, consistent with established negative feedback effects of adrenal steroids on CRH production [104]. Vasopressin can also be found in the majority of CRH-containing parvocellular neurons after adrenalectomy [104]; it has been suggested that this plasticity allows for synergy of stimulation of ACTH secretion by the two peptides [104]. CRH is also widely distributed throughout the brain in the cerebral cortex, limbic system, brain stem, and spinal cord [98–101]. It is also found extracerebrally, particularly in placenta; some investigators have also localized CRH in endocrine cells of the pancreas, gut and lung; in liver, adrenal [105], and testicular Leydig cells [106]. TRH

The first adenohypophysiotropic hypothalamic hormone identified was the tripetide TRH [17,18]. At first demonstrated to release TSH, TRH was rapidly also shown to release PRL. TRH binds to specific receptors on the thyrotroph and stimulates the transcription and secretion of TSH. TSH stimulates the thyroid gland to secrete primarily thyroxine, and triiodothyronine. The major source of triiodothyronine, however, is hepatic deiodination of thyroxine. These hormones act to regulate thermogenesis and protein synthesis. Immunocytochemical studies have localized TRH in the rat brain, in neuronal cell bodies of the preoptic nucleus, the parvocellular portion of the paraventricular nucleus, the perifornical region, dorsomedial nucleus, and basolateral hypothalamus [107,108]. TRH-positive terminals that contain dense core vesicles are found in high concentrations in the median eminence as well as in several of the hypothalamic nuclei, which also contain TRH-positive neuronal cell bodies. The tripetide is widely distributed throughout the brain and spinal cord, suggesting that it may act as a neurotransmitter in other areas [107,108]. Several effects of TRH not mediated by the pituitary gland have been demonstrated, including hyperthermia, behavioral excitation and the ability to reverse or prevent anesthesia from barbiturates and other depressants [109]. TRH has been reported in fetal and neonatal pancreatic islet cells [110] and gut endocrine cells [111], where it is thought to inhibit gastric secretion, gastric motility, and exocrine pancreatic secretion; it has also been localized in several human tumors [112]. GnRH

The second hypothalamic adenohypophysiotropic peptide to be characterized was originally called luteinizing hormone (LH)-releasing hormone (LRH) but was subsequently shown to release both gonadotropins, follicle stimulating hormone (FSH), and LH, by the pituitary gland [20–22]. The gonads are thereby stimulated to produce sex steroids and secondary sex characteristics. GnRH is also thought to stimulate sexual activity in experimental animals of both

12

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Hypothalamic–Pituitary Function

sexes, an effect that appears to be independent of its hypophysiotropic action [113]. GnRH has been localized in the hypothalami of several species [113–115]. In primates, GnRH neurons are located in large areas extending caudally from the preoptic region to the premamillary nucleus [96]. The neurons are bipolar or multipolar cells with multiple dendrites. The highest concentration of these cells is found in the mediobasal hypothalamus (infundibular and premamillary nuclei) and in the preoptic area. These neurons give rise to an infundibular GnRH pathway ending on the capillaries of the pituitary portal plexus as well as to a preopticoterminal tract projecting to the capillaries of the OVLT. In rodents, the majority of neuronal cell bodies are located in the septal preoptic regions and the anterior hypothalamus, and are particularly in the dorsal and medial septal nuclei, the nucleus of the diagonal band of Broca and anterior hypothalamic nuclei, and the OVLT. These neurons are usually elongated with eccentric nuclei and are unipolar. They project to the external zone of the median eminence as well as to the OVLT. Immunoelectron microscopy has documented localization of GnRH in dense core vesicles which measure 75–95 nm in diameter and are found in cell bodies as well as nerve endings [115]. Extrahypothalamic localization of GnRH includes several areas of the limbic system (septum, hippocampus, and olfactory bulb), the breast, and placenta [116]. Vasopressin and Oxytocin

Vasopressin, oxytocin, and their binding proteins, neurophysins I and II, are detected by immunohistochemistry in the perikarya of magnocellular neurons in the supraoptic (Fig. 1.6) and paraventricular (Fig. 1.7) nuclei [117,118]. Fibers emanating from these neurons converge towards the median eminence, pass caudally through its internal zone and end in dilated nerve terminals of the pars nervosa of

the pituitary known as Herring bodies (Fig. 1.8). The nerve fibers, their axonal swellings and the exceedingly large and characteristic swellings which can exceed 20 mm in diameter contain secretory granules of variable electron density and electron-lucent vesicles (Fig. 1.9). The secretory granules originate in the perikarya of the neurons and mature, growing from 60 to 220 nm in diameter, as they travel to the nerve endings [119,120]. It has been clearly shown that vasopressin and oxytocin are produced by different magnocellular neurons in both nuclei. Immunostaining for vasopressin is markedly increased after adrenalectomy in the external zone but not the internal zone of the rat median eminence [121,122]. Vasopressin is colocalized in some neurons with CRH and is additive with that substance in the regulation of ACTH release. Interestingly, it is found in high concentrations in portal blood draining to the anterior pituitary, suggesting that it may play a physiologic role in ACTH release [104]. A subset of oxytocin-containing neurons also may contain CRH. Immunoreactive vasopressin has been demonstrated in various other areas of the central nervous system as well as testis, ovary, uterus, adrenal, and thymus [123]. VIP

This 28-amino acid peptide, first isolated from intestine, has been localized in mammalian brain [124]. Immunoreactive fibers are present in high concentrations in the cortex, hippocampus, dentate gyrus, amygdala, nucleus of the stria terminalis, suprachiasmatic nucleus, and periaqueductal gray matter. Fibers containing immunoreactive VIP and its related PHI are also detected in areas that do not contain cell bodies. In the hypothalamus, VIP has been localized in the ventromedial nucleus, where it may affect appetite [56], in the suprachiasmatic nucleus, where it is thought to play a role in circadian rhythmicity, and in parvocellular neurons of the paraventricular nucleus [125]. It is known to modulate secretion of GH, PRL, and LH at the level of their hypothalamic regulation [126–128] and also stimulates PRL release directly [126]; some investigators have suggested that VIP is the physiologic PRL-releasing factor. After adrenalectomy, the number and intensity of VIP-containing small neurons in the paraventricular nucleus increase, suggesting a possible role for this substance in PRL regulation during stress. Cholecystokinin (CCK)

FIGURE 1.6. Perikarya and axonal processes of magnocellular neurons in the supraoptic nucleus contain strong immunopositivity for vasopressin. (Magnification ¥64)

This hormone, which was first discovered in the gut, was detected in the brain by immunocytochemistry [129–131]. CCK-containing cell bodies are prominent in the cerebral cortex and are also widely distributed throughout the olfactory and limbic systems. In the hypothalamus, they are found in several nuclei, including the magnocellular paraventricular and supraoptic nuclei. The midbrain also contains a striking concentration of CCK-immunoreactive cell bodies in the ventral tegmentum and raphe. This peptide modulates dopaminergic, noradrenergic, and opioid systems. It also inhibits feeding, probably at the level

Chapter 1

Functional Anatomy of the Hypothalamic Pituitary Axis

13

FIGURE 1.7. Adjacent to the third ventricle (V), magnocellular neurons in the paraventricular nucleus demonstrate strong immunoreactivity for vasopressin. (Magnification (a) ¥26; (b) ¥64)

FIGURE 1.8. The posterior lobe of the pituitary contains nerve fibers of the hypothalamohypophysial tract with axonal swellings and globular Herring bodies that stain with aldehyde thionin (a) and contain intense immunopositivity for vasopressin (b). (Magnification (a) ¥102; (b) ¥64)

of the ventromedial hypothalamus, and alters the pain threshold [132].

release, and to enhance GH release in response to GHRH in humans and rats [141], suggesting that hypothalamic galanin may modulate secretion of those hormones.

Galanin

Galanin is a 29-amino acid peptide that was originally isolated from intestine [133]. Immunoreactive galanin has been demonstrated in the central nervous system [134]; in hypothalamus, it colocalizes with dopamine and g-aminobutyric acid (GABA) [135]. It is also present in peripheral neural elements of the respiratory tract, gastrointestinal tract, pituitary, pancreas, urogentital tract, and adrenal medulla of humans and several mammalian species [136–139]. Galaninpositive neurons have been observed in several hypothalamic tumors [139]. Galanin is thought to act as a neurotransmitter although its functional roles have not been fully clarified. It has been shown to inhibit dopamine [140], thereby stimulating PRL

Gastrin

Gastrin is a gut peptide which is known to be produced in stomach and in numerous endocrine tumors of gut and pancreas. Specific antibodies have also localized gastrin in cerebral cortex, hypothalamus, and neurohypophysis [142,143]. Gastrin is known to modulate GH secretion at the hypothalamic level, either by reducing somatostatin release or by enhancing GHRH secretion [144]. Glucagon

Glucagon is expressed in the pancreatic islets and intestine, and has also been reported in other tissues, including thymus, thyroid, and adrenal medulla. A growing body of

14

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Hypothalamic–Pituitary Function

levels of NPY immunoreactivity are found in hypophysial portal blood. This peptide stimulates appetite when injected into the hypothalamus and appears to modulate the secretion of both LH and ACTH [150]. NPY-containing fibers also are known to contact TRH-synthesizing perikarya and neuronal processes in the paraventricular nucleus where it may mediate the neuroendocrine regulation of TRH [151]. Neurotensin

This 13-amino acid gut–brain peptide is found in the highest concentration in the ileum and hypothalamus [152]. In the rat, it is localized in the medial preoptic region, infundibular nucleus, paraventricular nucleus, and lateral hypothalamus with fibers most dense in the paraventricular and periventricular regions and in the median eminence. It has been shown to colocalize in neurons with catecholamines, possibly dopamine, in the periventricular and arcuate regions, and with CRH in the paraventricular nucleus. Some investigators have shown that it alters PRL release and blunts the response of that hormone to TRH [153]; it may also play a role in the regulation of GH and LH. Substance P

FIGURE 1.9. By electron microscopy, the normal human posterior lobe of pituitary contains numerous axonal processes filled with neurosecretory material (arrows). A large, dilated nerve terminal is the ultrastructural equivalent of the Herring body. (Magnification ¥7960)

evidence suggests that the brain is a site of glucagon biosynthesis. Neurons staining for glucagon and glucagon-like peptides have been detected in different regions of the brain including retina, hypothalamus, and medulla oblongata [145,146] and glucagon gene expression has been demonstrated in the brain stem and hypothalamus [147]. Intracerebral injections of glucagon produce dosedependent hyperglycemia, suggesting that this substance plays a role in the regulation of glucose metabolism centrally as well as peripherally [148]. Neuropeptide Y (NPY)

This 36-amino acid peptide is found by immunocytochemistry in a widely distributed pattern throughout the brain [149,150]. NPY-containing neurons are particularly numerous in the arcuate nucleus of the hypothalamus, striatum, hippocampus, and cortex. Nerve fibers are abundant in the paraventricular and suprachiasmastic nuclei and fibers are found in the internal zone of the median eminence. High

Substance P has been found in neurons in various regions of the brain. There are multiple substance P-containing afferents to the hypothalamus from the amygdala via the stria terminalis. Hypothalamic neurons containing this peptide are abundant in the arcuate, dorsomedial, ventromedial, and premamillary nuclei [152,154]. It is colocalized with vasopressin, is probably involved in pain perception within the limbic system, and may be secreted into the hypophysial portal vasculature where it is thought to affect blood flow. Substance P has been shown to alter GH regulation in vivo, but this is postulated to act via regulation of somatostatin neurons. It has variable effects on release of PRL, TSH, and LH. Bombesin/Gastrin-releasing Peptide (GRP)/Neuromedin

Bombesin was originally isolated from the skin of amphibians and stimulated great interest when it was shown to have potent biologic activity in mammals. Mammalian counterparts were soon discovered and given the names gastrin-releasing peptide and neuronedin. Bombesin-like immunoreactivity was found in neurons of the external zone of the median eminence near small blood vessels and colocalized with CRH [155]. Pharmacologic studies showed that bombesin can alter the release of adenohypophysial hormones including GH, PRL, and LH. The discovery of immunoreactivity for homologous peptides in hypothalamus suggests a possible role for these substances in the regulation of pituitary function. Calcitonin Gene-related Peptide (CGRP)

This 37-amino acid peptide is derived from the calcitonin gene by alternative messenger RNA (mRNA) processing.

Chapter 1

It is detected primarily in nervous tissue; CGRPimmunoreactive cell bodies are found in the supraoptic, paraventricular, and infundibular nuclei of the human hypothalamus [156]. CGRP inhibits GH release both in vitro and in vivo. It may also affect PRL release [157]. POMC-derived Peptides b-Endorphin

and other POMC-derived peptides are widespread in the central nervous system, their highest concentration being in the arcuate nucleus of the hypothalamus [158]. Nerve fibers containing b-endorphin and melanocyte-stimulating hormone-a (MSH-a) extend throughout many regions and innervate the median eminence, amygdala, preoptic area, ventromedial nucleus, and capillaries of the median eminence and portal system. There is another distinct and complex network of fibers containing the opiate methionine enkephalin (met-enkephalin). In addition to the multiple effects of these peptides on behavior, pain perception, immunomodulation, and many other functions too numerous to review here, they are also thought to modulate pituitary function [159], by inhibiting ACTH and LH release and stimulating PRL secretion. The effect on PRL is likely both direct and by reducing dopaminergic inhibition; the inhibition of LH appears to be only indirect via GnRH inhibition. The pituitary actions of endogenous opioids are complex, since they are themselves regulated by gonadal steroids and opioids may play a role in the negative-feedback effects of gonadal steroids on gonadotropins. Opioids also suppress the release of vasopressin and oxytocin, directly and possibly via noradrenergic regulation [160]. Cytokines

The close link between the neuroendocrine and immune systems has become widely recognized. Hormones are known to modulate the reactivity of immunocompetent cells and cytokines secreted by members of the immune system simulate secretion of hormones, most notably in the hypothalamus–pituitary–adrenal axis [161]. Interleukins are a family of peptides that mediate the immune response. Interleukin-1 (IL-1) is a monokine produced by macrophages in response to antigenic challenge. IL-1 stimulates ACTH and suppresses GH, LH and TSH secretion [162]. IL-1b immunoreactivity has been reported in neuronal fibers of the human brain, including the subfornical organ, stria terminalis, ventromedial nucleus of the hypothalamus, the posterior hypothalamus, and around the vessels of the median eminence. It is found in areas containing CRH-immunoreactive neurons and is known to increase CPH-mediated ACTH but is not found in the same synaptic vesicles as CRH. In fact, it is not released by depolarizing stimuli and it is not clear that IL-1b is even produced by neurons [161]. It also stimulates somatostatin biosynthesis in cultures of fetal rat brain [163], providing a

15

Functional Anatomy of the Hypothalamic Pituitary Axis

likely explanation for the in vivo reduction of GH and TSH secretion. Other interleukins are not known to be hypothalamic peptides but affect pituitary function. Interleukin-6 (IL-6) stimulates release of GH, PRL, and LH; tumor necrosis factor-a (TNF-a) and interferon-g also affect release of PRL and LH [164–166]. This information has significant neuroendocrinologic implications in the study of reproduction, autoimmunity, and other diseases. Growth Factors

A number of peptide growth factors, classified into several major families, are known to modulate cell proliferation. [167] Some are also known to affect hormone production (Table 1.1) and some are, in turn, modulated by hormones. Only a few have been identified in the hypothalamus and are thought to play a physiologic role in pituitary regulation; these include fibroblast growth factor (FGF) [168] and transforming growth factor-a (TGF-a) [169]. The former increases release of PRL and reduces basal GH; it also modifies the response of pituitary hormone release to TRH [170]. The latter has been localized by immunocytochemistry mainly in magnocellular neurons of the paraventricular and supraoptic nuclei of the hypothalamus. It is structurally homologous to epidermal growth factor (EGF) and may mediate some EGF effects on adenohypophysial production of GH, PRL, and TSH as well as cell proliferation [171].

Classification of Hypothalamic and Neurohypophysial Pathology Numerous pathologic processes involve the hypothalamus and neurohypophysis [46,47], giving rise to various symptoms, signs, and clinical syndromes. Among these, endocrine changes may be conspicuous. The local symptoms, such as visual defects, nerve palsy, and headache, may be secondary

Table 1.1. factors

Hormone regulation by pituitary-derived growth

Peptide

GH

PRL

EGF Activin FGF IGF-I IGF-II Insulin Endothelin Interleukin-1

Ø Ø

≠

Ø

FSH/LH

ACTH ≠

≠ ≠

Ø Ø Ø

TSH

≠

≠ ≠

≠ ≠

≠

GH, growth hormone; PRL, prolactin; TSH, thyroid-stimulating hormone; FSH, follicle-stimulating hormone; LH, luteinizing hormone; ACTH, adrenocorticotropic hormone; EGF, epidermal growth factor; FGF, fibroblast growth factor; IGF, insulin-like growth factor.

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to increased intracranial pressure or may be due to compression, invasion, injury, or destruction of tissue components. Involvement of certain parts of the hypothalamus can give rise to nonendocrine pathology, including abnormalities of appetite and temperature regulation. The endocrine sequela of hypothalamic disorders can be classified into two categories: (i) those giving rise to hypothalamic hormone deficiency; and (ii) those giving rise to hypothalamic hormone excess. The major pathologic processes causing these disorders are reviewed briefly and their features are fully discussed in ensuing chapters. Hypothalamic Hormone Deficiency

Insufficient secretion of hypothalamic adenohypophysiotropic hormones may be caused by destructive lesions of the hypothalamus. Lesions such as granulomas, meningitis or other inflammatory processes, pituitary adenomas, craniopharyngiomas, lymphomas, histiocytosis-X, metastatic carcinomas, craniocerebral trauma or hypoxic tissue injury may impair the synthesis of numerous hormones, including vasopressin, oxytocin, and the various adenohypophysiotropic hormones. These lesions give rise to true hypothalamic hypopituitarism. Similarly, lesions affecting the pituitary stalk, such as surgical or traumatic sectioning of the hypophysial stalk or its interruption by various pathologic processes, block the transport of the hypothalamic hypophysiotropic hormones from their sites of production in the hypothalamus to the anterior pituitary. It is obvious that any disconnection of the direct link between the hypothalamus and pituitary results in various degrees of hypopituitarism. Hypocortisolism, hypogonadism, and/or hypothyroidism have been demonstrated conclusively in patients with destructive diseases of the hypothalamus. Deficiency of GHRH usually results in growth retardation or in dwarfism [172]. Destructive lesions may manifest as growth retardation of hypothalamic origin in which patients respond to administration of GHRH. Idiopathic GH deficiency may be associated with other pituitary insufficiency and hypothalamic hormone abnormalities. Of particular interest is an autosomal recessive form of hypothalamic dwarfism due to failure to produce GHRH [173,174]; this disorder represents approximately 10% of cases of inherited growth retardation. The limited morphologic studies of patients with GHRH deficiency have documented that pituitary somatotrophs have normal morphology and GH content and administration of GHRH elicits a normal GH response [175]. Destructive lesions of the hypothalamus may impair the synthesis and/or release of dopamine and therefore may give rise to hyperprolactinemia [96]. Elevated blood PRL levels are also found in patients with disruption of, or damage to, the pituitary stalk. As a result of decreased transport of dopamine, lactotrophs are relieved from their tonic hypothalamic inhibition, leading to hyperprolactinemia. Hypothalamic hypothyroidism is usually not an isolated deficiency; it is most often associated with multiple hypo-

thalamic hormone deficiencies caused by organic hypothalamic pathology. Hypothalamic hypogonadism may be attributed to lack of GnRH or to nonpulsatile release of that hormone [176]. It may be an isolated defect of GnRH neurons [177]; in patients with hemochromatosis it is attributable to a combination of iron-induced damage to GnRH neurons and pituitary abnormalities [178]. Kallmann’s syndrome is an uncommon variant of hypothalamic hypogonadism which occurs in both men and women. The hypogonadism is associated with anosmia [179]. These deficits are due to defective development of the GnRH-secreting part of the hypothalamus associated with hypoplasia or aplasia of the olfactory area of the brain that can be documented by magnetic resonance imaging (MRI) [180]. Kallman’s syndrome is now known to be attributed to deletion or mutations of the KAL gene, located at Xp22.3; this gene encodes a 680 amino acid protein, anosmin, that has homology to other molecules implicated in neural development [181]. The pathology of the hypothalamus in this disorder has been thoroughly described in a histologic study in which abnormalities were found in various hypothalamic nuclei [182]; the lateral tuberal nuclei were underdeveloped and the subventricular nucleus exhibited neuronal hypertrophy. The number of pituitary gonadotrophs was markedly decreased as documented by immunohistochemistry and electron microscopy [182]. Detailed pathologic examination of the hypothalamus is yet to be reported in most patients with no major organic hypothalamic disease and apparently idiopathic deficiency of hypothalamic adenohypophysiotropic hormones. Diabetes insipidus is an uncommon but distinct clinical syndrome characterized by polyuria and polydipsia and results from vasopressin deficiency [46,47]. The extent of disease varies with the location of tissue destruction. Because vasopressin is transported through the pituitary stalk to the posterior lobe where it is stored in nerve endings, destruction of the posterior pituitary may interfere with storage and release of the hormone, resulting in its deficiency. This situation is usually transient and selective lesions of the posterior lobe usually cause only mild and temporary polyuria and polydipsia. Destruction high in the infundibulum results in permanent diabetes insipidus due to retrograde axonal degeneration and atrophy of neurons in the supraoptic and paraventricular nuclei [183]. Destruction of the supraoptic and/or paraventricular nuclei gives rise to permanent disease; approximately 90% destruction is required to cause significant and permanent disease [184], explaining why section low in the stalk, which creates atrophy of a minority of neuronal axons, causes only a transient disorder. Rare forms of hereditary idiopathic diabetes insipidus are usually unassociated with gross destruction of the hypothalamus [185]; detailed histologic studies reveal severe atrophy of neurons in the supraoptic and paraventricular nuclei in most patients but the etiology of these disorders remains unknown. In a few instances, neurons of the

Chapter 1

two magnocellular nuclei have been microscopically normal but devoid of vasopressin [186]. Some studies have suggested that this disorder may in some cases represent autoimmune destruction of vasopressin-producing neurons [187]. Oxytoxin deficiency, resulting from a decrease in the production of oxytocin, causes no major clinical abnormalities. Hypothalamic Hormone Excess

Hypersecretion of hypothalamic hormones, although rare, can result in endocrine symptomatology. Certain endocrine diseases characterized by hyperfunction of the anterior pituitary may be due primarily to adenohypophysiotropic hormone excess as a result of excessive secretion of hypothalamic releasing hormones. As yet, there is no evidence that hypersecretion of hypothalamic inhibiting hormones causes pituitary hypofunction. Acromegaly, a syndrome resulting from sustained GH hypersecretion, is almost always the result of a pituitary adenoma. Only one case of proven ectopic GH production has been reported [188]. More numerous are cases of production of GHRH by tumors that result in somatotroph hyperplasia or adenoma in the pituitary and produce clinical features of GH excess [87]. Extrahypothalamic GHRH production has been documented in endocrine tumors of lung, pancreas, thymus, medullary thyroid carcinoma, and pheochromocytoma. Primary tumors of the hypothalamus, called variously hypothalamic neuronal hamartomas, hypothalamic gangliocytomas and adenohypophysial neuronal choristomas, are lesions consisting of neurons with hypothalamic differentiation. Some of these tumors have been shown to contain GHRH and have been associated with pituitary somatotroph adenoma and acromegaly [189]. Several have been shown to be plurihormonal, containing GnRH, glucagon, gastrin, and somatostatin. Gigantism with pituitary GH excess has been described occasionally in the McCune–Albright syndrome [190]; this disorder is characterized by polyostotic fibrous dysplasia, pigmentary abnormalities and a variety of endocrine abnormalities, most commonly precocious puberty in female children. The pituitary may show mammosomatotroph hyperplasia that is attributed to activation of the signal transduction pathway generating cyclic AMP in these cells. These various manifestations are now known to be due to activating mutations of the Gs alpha gene (gsp mutations) [191,192]. Mammosomatotroph hyperplasia has also been reported as a cause of gigantism in the absence of McCune–Albright syndrome [193]. No proof of GHRH excess, either of hypothalamic or ectopic origin, has been offered in these patients; however, the pathogenetic role of GHRH in mammosomatotroph proliferation has been proven in mice transgenic for GHRH who have mammosomatotroph hyperplasia resembling that seen in these human disorders [194]. Cerebral gigantism (Sotos’ syndrome) has also been postulated to be a primary hypothalamic endocrinopathy. However, in patients with this

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disorder, hormone levels are normal and a morphologic study of the hypothalamus and pituitary showed no specific abnormality [47]. Cushing’s disease is known to be associated with pituitary corticotroph hyperplasia in some cases [195]; it has been suggested that the corticotroph hyperplasia may be secondary to excess production of CRH [196]. Ectopic production of CRH has been shown with increasing frequency and can cause Cushing’s disease; it must be considered in the differential diagnosis of that disorder [197,198]. In some cases, CRH has been elaborated in association with ACTH, suggesting autocrine regulation of some cases of ectopic ACTH syndrome. Rarely, a hypothalamic CRH-producing neuronal tumor has been reported in association with pituitary corticotroph hyperplasia or adenoma and Cushing’s disease [196,199–201]. Precocious puberty is an uncommon clinical syndrome which is in most instances idiopathic or attributable to androgen- or estrogen-producing neoplasms. Idiopathic precocious puberty is assumed to be hypothalamic in origin despite the absence of any anatomic lesion; less than 10% of cases are due to demonstrable hypothalamic pathology. Electroencephalographic abnormalities and behavioral disturbances have been observed in otherwise neurologically normal patients with this disorder [202]. Some patients respond to therapy directed against hypothalamic stimulation, but the disease may also be GnRH independent [203]. Structural lesions in and around the hypothalamus associated with precocious puberty fall into two groups. Some lesions actively participate in the production of precocious puberty by elaborating GnRH; these consist primarily of hypothalamic neuronal hamartomas which have been shown to contain GnRH in some cases [204,205]. The second group consists of hormonally inactive lesions which nonspecifically stimulate or inhibit hypothalamic centers involved in the regulation of sexual maturation. These are primarily lesions in the region of the posterior hypothalamus and include tumors of the pineal and hypothalamus, astrocytic gliomas, infections, hydrocephalus, head trauma, arachnoid cysts, and, occasionally, craniopharyngiomas. Precocious puberty is a prominent feature of the McCune–Albright syndrome [200]; in this disease, there is no demonstrable hypothalamic lesion and the patients may develop normal sexual function in adulthood. The hypothalamus has been implicated as the cause of some cases of hyperthyroidism but this has not been proven [206]. Vasopressin excess may cause water intoxication. The syndrome of inappropriate antidiuretic hormone (ADH) secretion (SIADH) may be due to idiopathic hypothalamic vasopressin excess or may be associated with various disease, including malignant tumors, most often bronchogenic carcinoma, and tuberculosis [207]. In the latter instances, this syndrome may be due to secretion of vasopressin or a similar peptide by tumoral or diseased pulmonary tissue [208,209]. In patients in whom no underlying cause is found, the

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idiopathic syndrome is likely due to posterior pituitary hyperfunction but no morphologic characteristics of this disorder have been described. The pathologic process in the brain may be in a number of areas; inhibitory osmoreceptors are found diffusely in the brain, as far away as the brain stem, and lesions can reduce the tonic inhibition of vasopressin, thereby increasing its release. Evidence of this widespread control is found in the increase in vasopressin release associated with congestive heart failure, cirrhosis, renal disease, brain trauma, positive pressure ventilation, and neurosurgical manipulation of the pituitary and hypothalamus. Age Related Changes in the Hypothalamus

Age related changes in the hypothalamus and neurohypophysis are few. The most striking is a morphologic curiosity, the accumulation of corpora amylacea, most often seen in the region of the supraoptic nuclei. The magnocellular nuclei are remarkably unaffected by age; immunohistochemistry shows no significant change in staining of vasopressin and oxytocin in older subjects [210]. The volume of the mamillary bodies is reduced but cell numbers appear normal [211]. Lipofuscin accumulation is commonly seen in the aging brain but does not appear to affect hypothalamic neuronal function. The suprachiasmatic nucleus decreases in volume and cell number with age [53]. The subventricular nucleus, in contrast, undergoes marked hypertrophy of its usually parvicellular neurons in postmenopausal women, as well as in starvation, posthypophysectomy, late pregnancy, and in patients suffering from prolonged postpartum hypopituitarism [58–64]. ADENOHYPOPHYSIS

Anatomy The adenohypophysis is composed of three parts, the pars distalis, the pars intermedia, and the pars tuberalis (Fig.

(a)

1.10). The pars distalis constitutes the largest portion of the gland; it is also known as the anterior lobe or the pars glandularis. The pars intermedia is rudimentary in the human pituitary; it is the vestigial posterior limb of Rathke’s pouch and is found in its underdeveloped form behind the medial cleft of the pituitary. It is also known as the intermediate lobe. The pars tuberalis is an upward extension of a few layers of adenohypophysial cells surrounding the external aspect of the lower hypophysial stalk; it is also known as the pars infundibularis. The adult human pituitary is bean shaped, bilaterally symmetrical, and measures approximately 13 mm transversely, 9 mm anteroposteriorly, and 6 mm vertically. It weighs approximately 0.6 g. The anterior lobe comprises about 80% of the pituitary. The pituitary of women is somewhat larger than that of men; moreover, the pituitary is heavier in multiparous than in nulliparous women. The increased size is attributable to the marked hyperplasia of the pituitary during pregnancy and lactation, which may increase the weight to 1 g or more; postlactational involution occurs but the gland does not return to its prepregnancy size. A slight to moderate weight reduction is seen with advancing age. The hypophysis is found within the sella turcica where it is lined by dura mater, a dense layer of connective tissue. The uppermost portion of the adenohypophysis is covered by the diaphragma sellae, a reflection of the dura that constitutes the roof of the sella turcica. The sellar diaphragm has a small central opening for the hypophysial stalk and protects the pituitary from the pressure of cerebrospinal fluid (CSF). Defective development or absence of this structure may lead to the so-called empty sella syndrome in which the pituitary is compressed by CSF pressure. The increased pressure results in enlargement of the sella turcica and flattening of the pituitary, which may be compressed to a thin layer of tissue at the bottom of the sella turcica. This lesion is usually unassociated with functional

(b)

FIGURE 1.10. (a) Sagittal section of an adult human pituitary obtained at autopsy illustrates the adenohypophysis (A) and neurohypophysis (N) attached to the pituitary stalk. (b) Horizontal cross-section of an adult human autopsy pituitary allows accurate identification of the neural lobe (N) and the anterior lobe (A) with subtle distinction between the two lateral lobes and the median wedge. The cystic remnants of Rathke’s cleft that represent the pars intermedia in humans are indistinct (arrow head).

Chapter 1

hypophysial abnormalities [206] but is important in differential diagnosis. Other anatomic variations in the shape of the hypophysis, the size and width of the sellar diaphragm, and relation to pituitary structures have been reported [207]. Minor changes have no endocrine significance.

Vascular Supply The blood circulation of the human hypophysis has major implications for the regulation of adenohypophysial hormone secretion [48–51]. The gland receives blood through the hypophysial portal circulation which carries the hypothalamic stimulatory and inhibitory hormones from the infundibulum to adenohypophysial cells. As described above (see Fig. 1.2), the branches of the superior hypophysial arteries penetrate the stalk, form a superficial network of vessels (external plexus) and give rise to a mesh of capillaries and gomitoli (internal plexus). The latter presumably regulate blood flow, thereby affecting transport of hypothalamic hormones to the adenohypophysis. The long portal vessels that arise from the infundibular plexuses and the short portal vessels that originate in the lower pituitary stalk and posterior lobe enter the pars distalis carrying adenohypophysiotropic hormones; the former carry 70–90% of the blood flow while only 10–30% originates in the short portal vessels. The trabecular or loral arteries transport blood to the adenohypophysis via the neural lobe, but, in addition, some arterial blood is directed to the adenohypophysis via two branches of the inferior hypophysial artery: (i) the capsular artery, which serves the connective tissue of the pituitary capsule and penetrates to the superficial cell rows of the adenohypophysis; and (ii) the artery of the fibrous core. In some individuals, the middle hypophysial artery may vascularize the adenohypophysis directly [212]. The volume of veins leading away from the adenohypophysis is considerably smaller than that of portal vessels entering the gland. The meager venous connections drain adenohypophysial and neurohypophysial blood to the cavernous sinus. It has been shown that the short portal vessels also serve as efferent channels, giving rise to reversal of blood flow in the neurohypophysial vascular bed. This implies that secretory products of the adenohypophysis may also enter the neurohypophysis and, by reverse flow to the median eminence, play a role in the regulation of hypothalamic factors via blood flow [50,51]. Pituitary capillaries are lined by fenestrated endothelium with a thin subendothelial space. Hormones released by adenohypophysial cells must therefore pass through the basement membrane of their cell of origin, capillary basement membrane, subendothelial space, and the endothelial cell layer to reach the bloodstream.

Nerve Supply The adenohypophysis has no direct nerve supply, apart from small sympathetic nerve fibers which are associated with and

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presumably innervate capillaries [195]. In some species, the intermediate lobe has rich innervation [213]. Thus, although neural connections may affect blood flow to the adenohypophysis, it is unlikely that they are involved in the regulation of adenohypophysial hormone secretion, which is predominantly neurohumoral.

Embryology All three portions of the adenohypophysis are assumed to derive from Rathke’s pouch, an endodermal pouch of the primitive oral cavity. At the third week of gestation, endoderm from the roof of the stomodeum thickens and begins to invaginate; by 5 weeks, Rathke’s pouch is a long tube with a narrow lumen and a thick wall composed of stratified cuboidal epithelium. By 6 weeks, the connection with the oropharynx is totally obliterated and Rathke’s pouch establishes direct contact with the downward extension of the hypothalamus that gives rise to the infundibulum. The two tissues are enclosed by the cartilage anlage of the sphenoid bone, separating them from the stomodeum; the sella turcica is formed by 7 weeks [66]. It was suggested that Rathke’s pouch arises from the ventral neural ridge in the pharyngeal region, thus sharing with the hypothalamus and posterior pituitary a common neuroectodermal origin [214–217]. Some features of adenohypophysial cells suggest that they are members of the APUD (amine precursor uptake and decarboxylation) cell system. The use of avian allografts, biologic markers, and serial sections of early chick embryos has provided indirect evidence for this theory; however, further proof is required for its validation. As the cells of Rathke’s pouch proliferate, the anterior portion forms the pars distalis and pars tuberalis whereas the posterior wall lies in direct contact with the posterior lobe anlage and becomes the pars intermedia [218,219]. The growth of the anterior limb extends laterally and follows a triradiate pattern; the lateral borders become the lateral wings of the adult gland and the midline portion becomes the anteromedial “mucoid wedge.” By midgestation, the medial cleft becomes a residual lumen and growth of the pars nervosa reverses the convexity of the posterior wall of the cleft to a concave structure. The border between Rathke’s pouch and the pars nervosa becomes indistinct; it consists of remnants of the obliterating lumen, and a few cystic cavities lined by cuboidal or columnar epithelium. This represents the rudimentary pars intermedia of the human hypophysis. The pituitary grows rapidly in early fetal life: the mean weight at 10–14 weeks of gestation is 3 mg, at 25–29 weeks 50 mg, and at term approximately 100 mg [218]. The pituitary portal vascular system begins to form before 7 weeks of gestation, and by 12 weeks the anterior pituitary and median eminence are well vascularized. Portal vessels are recognized at 11.5–14 weeks, are well developed by 15–16 weeks, and are fully established by 18–20 weeks [71,72].

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The first cell type to develop in the human fetal pituitary is the corticotroph [219–221]; at 6 weeks of gestation, cells with ultrastructural features of differentiating corticotrophs are found, and by 7 weeks, ACTH immunoreactivity is detectable. At 8 weeks of gestation, somatotrophs are identified by ultrastructural criteria and contain intense GH immunoreactivity. a-Subunit of the glycoprotein hormones is found in cells which have features of the glycoprotein hormone cell line; differentiated thyrotrophs and gonadotrophs are found subsequently at 12 weeks when b-subunits of those hormones are immunolocalized. Gonadotrophs show sexual dimorphism in the fetus; the differences are most marked from 15–25 weeks when this cell type is more numerous in female glands. Throughout gestation, LH-containing cells predominate in male glands, unlike the more equal distribution of LH- and FSHcontaining cells in female tissues [220]. Lactotrophs are the last cells to differentiate in the human fetal pituitary. PRL is found only at 12 weeks of gestation and is localized in mammosomatotrophs, bihormonal cells which appear to be the sole source of PRL until 24 weeks of gestation [221]. Differentiated lactotrophs are found after that time and undergo a striking hyperplasia in the late third trimester, analogous to that seen in late gestation and lactation in the pituitary of the mother. Novel transcription factors that play a role in anterior primordial development are being identified at a rapid pace. Many of these are implicated in early pituitary organogenesis, including the bicoid-related pituitary homeobox factor Ptx1 [222], Pituitary homeobox factor 2 (Ptx2), structurally related to Ptx1 [223], two members of the Lhx gene family, a group of LIM homeobox genes, Lhx3 and Lhx4 [224] and P-LIM, another LIM homeobox protein transcription factor that is selectively expressed in the pituitary with highest levels at the early stages of Rathke’s pouch development [225]. Another early marker of pituitary differentiation is the Rathke’s pouch homeobox (Rpx) protein which is identified in the pituitary primordium prior to the onset of known pituitary hormone production [226]. The Prophet of Pit-1 (PROP-1) is a paired-like homeodomain protein that is expressed early in pituitary development. It induces Pit-1 expression and plays a role in downregulation of Rpx [227,228]. Inactivating mutations of PROP-1 have been identified as the cause of Pit-1 deficiency in Ames dwarf mice [227] and in humans with combined pituitary hormone deficiency [229,230]. Id, a member of the helix-loop-helix (HLH) family of transcription factors is also found early in development and in some pituitary tumor cell lines but is decreased or absent in differentiated cells [231]. Its role in pituitary cytodifferentiation remains unclear. The molecular factors that determine hormone production have now been identified as transcription factors that target specific hormone genes. These factors have clarified three main pathways of cell differentiation [232,233]. ACTHproducing corticotrophs are determined by a novel T box

factor in cooperation with Ptx1 and corticotropin upstream transcription-binding element (CUTE) proteins including neuroD1/beta2 [234,235]. Bihormonal gonadotrophs require expression of steroidogenic factor (SF)-1 [236,237]. Pit-1 [238] directs differentiation of a complex family of cells that can mature into somatotrophs, mammosomatotrophs, lactotrophs or thyrotrophs [239–241] with the additional expression of estrogen receptor (ER) a [242], which enhances PRL secretion, or thyrotroph embryonic factor (TEF) which stimulates TSH-beta production [243]. The recognition of these molecular determinants of adenohypophysial cytodifferentiation has clarified the patterns of plurihormonality which have been recognized in pituitary adenomas and provide a framework for classification of these tumors. In rodents and humans, differentiation and/or maintenance of somatotroph, lactotroph and thyrotroph phenotypes are dependent on expression of a functional pit-1 gene; mutations in the pit-1 gene result in hypopituitarism [244–247] and hypoplasia of somatotrophs, lactotrophs and thyrotrophs [244]. An interesting observation is that Pit-1 mRNA and protein are highly expressed during human pituitary development at 17–19 weeks, when GH levels are extremely high, and near term when there is proliferation of lactotrophs [248]. These data suggest that Pit-1 plays an important role not only in the differentiation process, but also in the regulation of hormonal activity and possibly also of cell proliferation. Gonadotroph differentiation likewise requires SF-1; mice with disruption of SF-1 fail to develop pituitary gonadotrophs [236] as well as a ventromedial nucleus of the hypothalamus and steroidogenic glands [249].

Functional Anatomy In contrast with the hypothalamus, the cell types of the adenohypophysis are highly characterized with respect to structure and function. It is notable that with the exception of ACTH and a-subunit of the glycoprotein hormones, ectopic production of pituitary hormones is rare. Somatotrophs

Approximately 50% of the cells of the adenohypophysis produce GH. Most of these are located in the lateral wings of the anterior lobe; scattered somatotrophs are found in the median wedge [195]. These cells can usually be identified by light microscopy as medium-sized acidophilic cells that stain with eosin, phloxin, and orange G. They are spherical or oval in shape, with central, spherical nuclei. Immunohistochemistry reveals intense positivity for GH distributed throughout the cytoplasm [38] and for Pit-1 in the nucleus [221,250]. Occasionally, smaller cells contain GH positivity in a globular structure which represents the Golgi complex; these may be sparsely granulated, actively secreting cells. In situ hybridization localizes GH mRNA to both densely granulated acidophils and to occasional chromophobes (Fig. 1.11). By electron microscopy, somatotrophs are spher-

Chapter 1

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FIGURE 1.11. In situ hybridization localizes growth hormone messenger RNA in the normal pituitary in large cells with acidophilic cytoplasm and in occasional chromophobes. (Magnification ¥102)

ical or oval cells with centrally located spherical nuclei and cytoplasm of relatively low electron density [40]. The prominence of rough endoplasmic reticulum and Golgi regions varies with the secretory activity of the cell; active cells generally have well-developed, lamellar rough endoplasmic reticulum, and a large Golgi complex, whereas less active ones tend to have less conspicuous synthetic organelles. In the majority of somatotrophs, the cytoplasm is occupied by spherical, evenly electron-dense secretory granules which store hormone (Fig. 1.12). The limiting membrane of the granules is tightly opposed. The secretory granules range widely in size from 150 to 800 nm in diameter with an average of 350–500 nm. Secretory granules may be found accumulated at the cell periphery but granule extrusions are not detected. Ultrastructural immunocytology confirms the presence of GH in secretory granules and within the Golgi region of actively secreting cells. A subset of these cells has been recognized to contain a-subunit of glycoprotein hormones [40]. The incidence, distribution, morphology, and hormone content of somatotrophs are remarkably constant in the human pituitary. They do not appear to be affected by age, sex, various disease states, or drug therapies. Even in glands containing GH-producing adenomas the nontumorous somatotrophs show no evidence of suppression and cannot be distinguished from those in normal glands [40]. An exception to this morphologic uniformity is the case of long-standing hypothyroidism in which some degranulation of somatotrophs may occur; this change is much less marked than the almost complete degranulation of somatotrophs due to hypothyroidism which has been documented in rodents [250]. In pituitary adenomas, somatotrophs display a striking dimorphism [195]. Despite the morphologic differences between densely granulated and sparsely granulated soma-

FIGURE 1.12. A nontumorous somatotroph contains short profiles of rough endoplasmic reticulum (arrow head), a moderately developed Golgi complex (G) and numerous spherical, evenly electron-dense secretory granules with tightly fitted limiting membranes. (Magnification ¥5500)

totroph adenomas, both types have similar clinical features with elevated blood GH levels and acromegaly. Although the GH concentration in the tumor is proportional to the degree of granularity, neither of these correlate with blood GH levels. The morphine variant does, however, predict response to octreotide therapy [251]. Densely granulated somatotroph adenomas are composed of intensely acidophilic cells which contain strong immunoreactivity for GH throughout their cytoplasm. The tumor cells resemble resting nontumorous somatotrophs, with welldeveloped rough endoplasmic reticulum, prominent Golgi complexes and numerous spherical, evenly electron-dense secretory granules which measure 300–600 nm in diameter. These tumors may contain immunoreactivity for prolactin and/or a-subunit of glycoprotein hormones. The presence of these substances and GH in the same secretory granules has been documented by ultrastructural immunocytology using the double immunogold technique [252]. The use of keratin immunocytochemistry distinguishes these cells from

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FIGURE 1.13. A sparsely granulated adenomatous somatotroph harbors a characteristic juxtanuclear fibrous body composed of intermediate filaments that trap smooth endoplasmic reticulum, Golgi saccules (arrowheads), centrioles (arrows) and pleomorphic forming secretory granules. (Magnification ¥11390)

other somatotroph adenomas. Densely granulated cells have perinuclear low molecular weight cytokeratin filaments. Sparsely granulated somatotroph adenomas are composed predominantly of chromophobic or only slightly acidophilic cells which may show considerable nuclear pleomorphism. The nuclei are generally concave and binucleate; multinucleate cells are easily found. By light microscopy, spherical light areas can be seen adjacent to the nuclei; these correspond to the fibrous bodies seen by electron microscopy and are known to contain immunoreactive keratin [254]. The application of cytokeratin staining allows recognition of fibrous bodies that characterize this tumor [253]. GH positivity is found generally in the juxtanuclear Golgi region, while secretory granules, which are few, can be seen usually only with oil immersion. By electron microscopy, this tumor has a highly characteristic ultrastructure which differs significantly from that of nontumorous somatotrophs (Fig. 1.13). The tumor cells are irregularly shaped with numerous indentations and have lobulated pleomorphic and eccentrically located nuclei. The globular cytoplasmic

fibrous body, found in up to 95% of cells in sparsely granulated somatotroph adenomas, consists of intermediate-sized filaments that accumulate in the Golgi region, close to the concave side of an indented eccentric nucleus. The nearly perfectly globular structure traps tubular smooth endoplasmic reticulum, Golgi saccules, centrioles, mitochondria, lysosomes, and secretory granules. The secretory granules in the cells are scattered throughout the cytoplasm and are smaller than in densely granulated cells, measuring 100 – 250 nm in diameter. No exocytoses are seen. GHRH has been shown to stimulate proliferation of GH-containing cells in vitro during long-term incubations [255]. The effects of chronic exposure to GHRH in humans can be deduced from the study of pituitaries of patients with tumors producing GHRH [40,87]. These patients have marked somatotroph hyperplasia; the somatotrophs are generally large with very well-developed rough endoplasmic reticulum profiles and large Golgi complexes. The hyperplastic cells have numerous large electron-dense secretory granules characteristic of nontumorous cells; sparsely granulated cells with fibrous bodies are generally not seen. Although short-term exposure to GHRH is known to deplete the GH pool [256] and to convert densely granulated cells to more sparsely granulated forms [257], cells of densely granulated somatotroph adenomas chronically exposed to GHRH in vitro show an increase in the cytoplasmic volume densities of rough endoplasmic reticulum and Golgi complexes and a decrease in cytoplasmic volume density of secretory granules. However, they do not assume the characteristics of sparsely granulated cells and do not contain fibrous bodies [258]. Sparsely granulated adenomas undergo similar alterations during long-term exposure to GHRH in vitro and retain their characteristic fibrous bodies [258]. Although GHRH stimulates somatotroph proliferation in patients with GHRH-producing tumors, the hyperplastic adenohypophysial cell population in mice transgenic for this hormone is dominated by mammosomatotrophs as well as somatotrophs [194]. The presence of mammosomatotrophs may be attributed to exposure to GHRH excess in these animals during fetal life, at which time mammosomatotrophs are numerous, in contrast to the usual onset of tumoral GHRH excess in adulthood. Further studies are required to clarify the complex regulation of somatotroph and mammosomatotroph differentiation and proliferation by GHRH. The morphologic effects of somatostatin exposure on nontumorous somatotrophs has not been documented. The use of somatostatin analogs, such as SMS 201–995 (Sandostatin or Octreotide) in the therapy of acromegaly has allowed morphologic study of the effects of that substance on somatotroph adenoma cells. Both in vivo [259–261] and in vitro [262], somatostatin and its long acting analogs induce inconsistent morphologic alterations. In some tumors, there is no change, while in others, cell size is reportedly reduced. In some cases, there is an increase in the cytoplasmic volume density of lysosomes unassociated with significant alteration

Chapter 1

of other subcellular organelles. A few studies have indicated that these substances may induce fibrosis; others have found vascular changes which may cause cell necrosis and tumor shrinkage but direct cytotoxic effects have not been documented in vitro. No inhibition of hormone synthesis has been identified, as indicated by reduction in the cytoplasmic volume density of rough endoplasmic reticulum or Golgi complexes; this suggestion confirms molecular studies which have shown no suppression of mRNA in somatostatinexposed nontumorous adenohypophysial cells [263] or tumors [264]. The morphologic studies suggest that somatostatin suppresses hormone release and may stimulate lysosomal degradation of stored hormones. There is, however, differential response to octreotide in vivo by the two somatotroph adenoma types; densely granulated tumors exhibit greater reduction of growth hormone secretion than do sparsely granulated tumors [251]. Lactotrophs

Lactotrophs, or PRL cells, represent approximately 15% of the cells of the adenohypophysis. However, the number of these cells shows wide variation, related to age, sex, and parity in women. In adult men and nulliparous women they constitute approximately 9% of adenohypophysial cells, whereas in multiparous women they represent up to 31% of the cell population [266]. PRL cells are randomly distributed throughout the anterior lobe but are most numerous in the posteromedial portions, with large numbers extending to the posterolateral aspect [195]. Using conventional stains, PRL cells are acidophils that are indistinguishable from somatotrophs; however, Herlant’s erythrosin and Brookes’ carmoisine allow selective visualization of densely granulated lactotrophs, although these techniques are inconsistent and are not

FIGURE 1.14. Immunocytochemistry identifies prolactin diffusely in the cytoplasm of polyhedral and elongated densely granulated cells (arrow), and in the juxtanuclear globular Golgi region of sparsely granulated cells (arrow head). (Magnification ¥102)

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sufficiently sensitive to detect sparsely granulated forms. Immunohistochemistry reveals larger numbers of PRLcontaining cells among which two populations can be distinguished [38] (Fig. 1.14). Densely granulated, polyhedral, or elongated cells are frequently found close to capillaries randomly distributed throughout the anterior lobe; they have abundant cytoplasm almost completely filled with dense granular positivity corresponding to secretory granules. Sparsely granulated cells, in contrast, are more numerous and are found predominantly in clusters at the posterolateral portion of the gland. They are elongated or angular cells with long cytoplasmic processes and strong immunoreactivity for PRL in a juxtanuclear globular Golgi complex. It has been postulated that the densely granulated cells store PRL, whereas the sparsely granulated cells are actively secreting forms. In addition to nuclear Pit-1 reactivity these cells may also exhibit staining for estrogen receptor a [265a,265b]. By electron microscopy, the two cell types are readily distinguishable [40] (Fig. 1.15). Densely granulated cells are

FIGURE 1.15. A nontumorous lactotroph has abundant cytoplasm with highly developed rough endoplasmic reticulum in parallel arrays (*) and few, small secretory granules. (Magnification ¥6080)

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rare in the adult pituitary but are more common in childhood and adolescence. They have ovoid or elongated cell bodies, well-developed rough endoplasmic reticulum at the cell periphery and a spherical or oval Golgi complex containing a few forming granules. The cell cytoplasm is almost completely filled with spherical, oval, or irregularly shaped large granules that have evenly electron-dense cores and measure up to 650 nm in diameter. Granule extrusions are seen occasionally, usually on the cell surfaces overlying basement membrane. The majority of lactotrophs in the adult gland are sparsely granulated, elongated or polygonal cells which may have multiple cell processes extending from the center of acini to the basement membrane and intimately surrounding gonadotrophs. They contain richly developed rough endoplasmic reticulum found in parallel arrays and occasionally forming concentric structures known as “Nebenkern” formations. The Golgi apparatus is prominent and contains pleomorphic immature secretory granules. Scattered secretory granules are fewer and smaller than in storing cells; the diameters range from 150 to 250 nm. Granule extrusions are common and are found not only at the basal cell surface but also on the lateral cell borders, distant from capillaries and basement membranes; the term “misplaced exocytosis” is used to designate this form of granule extrusion. Ultrastructural immunocytology localizes PRL in secretory granules of both cell types; cloudy positivity can also be found in the Golgi region of sparsely granulated forms. In the fetus [220] and newborn as well as during pregnancy and lactation [266,267] there is marked hyperplasia of PRL cells which has been attributed to stimulation by estrogens. During pregnancy and lactation the weight of the pituitary gland may increase to more than 1 g and almost 50% of the total pituitary cell population is composed of PRLcontaining cells. Exogenous estrogens are known to induce only mild hyperplasia of lactotrophs in the human pituitary; the increased cell population is composed predominantly of the small sparsely granulated cell type [268a]. With sustained stimulation of PRL synthesis due to decreased dopamine inhibition or increased estrogen levels, there is a progressive increase in cytoplasmic volume and accumulation of highly organized rough endoplasmic reticulum, enlarged Golgi, numerous forming granules, and few small stored granules [40]. In animals lacking the dopamine D2 receptor, there is extensive lactotroph hyperplasia that progresses to adenoma [268b]. Suppressed lactotrophs are found in some pituitary glands containing PRL-producing adenoma or in patients treated with dopamine agonists. These inactive cells have a reduced nuclear volume, irregular indented nucleus with coarse clumped heterochromatin, small cytoplasm with poorly developed organelles, and secretory granules which measure 50–300 nm; the granules have an increased relative cytoplasmic volume due to the markedly reduced total cytoplasmic volume. Occasional exocytoses may be found at the basal cell surface. These suppressed lactotrophs are identified by immunocytology at the electron microscopic level; they

cannot be characterized on the basis of their ultrastructural features alone, which are not unlike those of null cells. PRL cell adenomas also occur in densely and, more commonly, in sparsely granulated varieties [195]. There is no correlation between morphology and blood PRL levels, age, or sex of the patient. The cells resemble the densely and sparsely granulated cells of the nontumorous pituitary. Effects of dopamine agonists on the cells of lactotroph adenomas include significant reduction of cytoplasmic, nuclear, and nucleolar areas [269]. There is also a marked reduction in the cytoplasmic volume densities of rough endoplasmic reticulum and Golgi complexes, whereas secretory granules increase in size and cytoplasmic volume density. The size reduction of bromocriptine-treated prolactinomas is therefore attributable to reduction of individual cell size rather than to a decrease in cell number. Protracted therapy may result in fibrosis which can adversely affect surgical outcome [270]. Mammosomatotrophs

The existence of bihormonal cells containing both GH and PRL in the nontumorous human pituitary has been recognized only in the last few years [40]. This cell type was initially identified in pituitary tumors [195]; it was subsequently found to be the cell of origin of PRL in the fetal pituitary during the first half of gestation [221]. Mammosomatotrophs are thought to be randomly distributed throughout the anterior lobe. They cannot be recognized using conventional histologic techniques; they are acidophils and are indistinguishable from somatotrophs. Immunohistochemistry reveals intense GH content within these densely granulated, polyhedral cells. PRL content is also identified but staining is usually less intense. Nuclear Pit-1 and ER staining is usually strong [239,240,265a,265b] and these cells often contain a-subunits [253]. By electron microscopy these cells resemble densely granulated somatotrophs (Fig. 1.16a). They are fairly large, polyhedral cells with ovoid or slightly irregular nuclei. They have abundant electron-lucent cytoplasm which contains well-developed rough endoplasmic reticulum and a prominent Golgi apparatus which often harbors numerous immature secretory granules. The mitochondria vary from ovoid to rod-shaped; they have a light matrix and lamellar cristae. The distinctive feature of these cells is their unique population of secretory granules. Some are small, spherical, or slightly ovoid, electron-dense granules which measure 150–400 nm; they have tightly fitting limiting membranes. The larger granules are irregular, often extremely elongated structures which can measure from 350 to 2000 nm; they contain secretory material of variable electron density and are bound by a loosely fitting membrane. These secretory granules frequently show misplaced exocytosis with fusion of the limiting membrane to the cell membrane. Unlike the granule extrusions of lactotrophs, the contents that are emptied into the intercellular space persist and retain electron density. Ultrastructural

Chapter 1

(a)

Functional Anatomy of the Hypothalamic Pituitary Axis

25

(b)

FIGURE 1.16. (a) By electron microscopy, mammosomatotrophs resemble densely granulated somatotrophs but have numerous conspicuous large secretory granules of irregular shape and variable electron density, as well as the hallmark of prolactin secretion, the misplaced exocytosis (arrow). (Magnification ¥7300) (b) For the definitive evidence of bihormonal differentiation of mammosomatotrophs, ultrastructural immunocytology localizes both growth hormone (15 nm gold particles) and prolactin (40 nm gold particles) to the same cell, often within the same secretory granule. (Magnification ¥20160) (b) From Felix et al. [271]

immunocytology using the double immunogold technique documents the presence of both GH and PRL in a single cell, frequently within the same secretory granule (Fig. 1.16b). Hyperplasia of mammosomatotroph cells has been identified as a cause of gigantism of early onset [193] and has been associated with acromegaly and hyperprolactinemia in the McCune–Albright syndrome [190]. Although the pathogenesis of this lesion in human patients remains unclear, an animal model for this disorder is provided by giant mice transgenic for GHRH in whom chronic exposure to excess GHRH causes mammosomatotroph hyperplasia [194]; continued long-term GHRH stimulation leads to the development of mammosomatotroph adenoma in old animals [272]. In human patients, tumors composed of mammosomatotrophs are more common than assumed in the past; they are slowly growing adenomas most often found in young patients with gigantism and mild hyperprolactinemia [195,252]. Corticotrophs

A single cell type in the human pituitary is responsible for the production of the POMC molecule and its various derivatives, including ACTH, MSH, lipotropic hormone (LPH), and endorphins [195]. Corticotrophs comprise approximately 15–20% of the adenohypophysial cell popu-

lation. The vast majority of these cells are found in clusters in the central mucoid wedge of the adenohypophysis; occasional scattered cells are also found in the lateral wings of the anterior lobe. Corticotrophs are also the predominant cell type in the poorly developed intermediate lobe of the human pituitary where they are found scattered in follicular structures. By light microscopy these medium-sized cells have varying degrees of cytoplasmic basophilia and stain strongly with PAS; the affinity is attributed to the carbohydrate moiety present in ACTH precursors. They also stain with lead hematoxylin. The most reliable method of identifying corticotrophs is the immunoperoxidase technique which reveals strong granular cytoplasmic positivity for ACTH. The presence of a large unstained perinuclear lysosomal vacuole known as the “enigmatic body” may be helpful in identifying these cells. Corticotrophs can also be identified by in situ hybridization which localizes the POMC mRNA [273]. Immunocytologic studies have indicated that almost all corticotrophs contain immunoreactive MSH, LPH, endorphins and other fragments of the POMC molecule which are derived by differing posttranslational processing [158]. By electron microscopy (Fig. 1.17), corticotroph cells are oval or slightly angular medium-sized cells with spherical or oval eccentric nuclei and a spherical nucleolus which is

26

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Hypothalamic–Pituitary Function

FIGURE 1.18. In the pituitary of a patient treated with high doses of glucocorticoids, corticotrophs with Crooke’s hyaline change have glassy cytoplasm and their periodic acid–Schiff (PAS) positive secretory material is pushed to the rim of the cell; the large juxtanuclear globule (arrow head) is the enigmatic body. (PAS stain; magnification ¥256)

FIGURE 1.17. The nontumorous corticotroph has widely dispersed short rough endoplasmic reticulum membranes, a prominent Golgi complex (G) and numerous secretory granules of variable size, shape and electron density. The two highly characteristic features of this cell type are the perinuclear and cytoplasmic bundles of intermediate filaments (arrows) and the large lysosomal enigmatic body (EB). (Magnification ¥5150)

usually attached to the nuclear membrane [40]. The cytoplasm has varying electron density and is frequently moderately electron-opaque. The rough endoplasmic reticulum is moderately developed and takes the form of widely dispersed membranes. Numerous free ribosomes can be found. The Golgi apparatus is spherical or flattened and is often displaced by the “enigmatic body.” This large structure is membrane-bound and has an electron-dense periphery that exhibits acid phosphatase activity, confirming its lysosomal nature [274]. Mitochondria are spherical or oviod, with lamellar or tubular cristae and a moderately electron-dense matrix. Variable numbers of intermediate filaments of the cytokeratin type [275], previously described as type I microfilaments, are found in small bundles usually adjacent to the nucleus; they measure about 7 nm in width and show no periodicity. They vary considerably in amount and are not numerous under physiologic conditions. The secretory granules are usually numerous and extremely variable in size,

shape, and electron density. They may be spherical, flattened, dented, heart shaped, or tear-drop shaped. They vary in electron density and may be 150–700 nm in diameter, most measuring 150–400 nm. While secretory granules may be found lined up along the cell membrane, exocytosis is not described in this cell type. Immunoelectron microscopy identifies the various POMC-derived peptides in the secretory granules of corticotrophs; however, there is no evidence that the morphologic differences of granule populations reflect their content [38]. Corticotrophs are the first cell type to differentiate in the fetal pituitary [220,221]; however, they are found in significantly decreased numbers in anencephaly [276]; it has been suggested that after autonomous differentiation, they are dependent on hypothalamic factors for normal growth and development. Their numbers do not vary with age or changes in the hormonal environment; however, they do develop specific morphologic features that reflect changes in endocrine homeostasis. In the human pituitary exposed to glucocorticoid excess due to administration of exogenous corticosteroids or any cause of endogenous glucocorticoid hypersecretion (including ectopic secretion of ACTH), corticotrophs undergo a distinctive morphologic alteration known as Crooke’s hyaline change [195]. By light microscopy, the cells accumulate a glassy, homogeneous, slightly acidophilic substance in the cytoplasm; the PAS positivity and ACTH immunoreactivity, corresponding to secretory granules, are displaced to the perinuclear rim and the periphery of the cell [38] (Fig. 1.18). The hyaline material does not contain immunoreactive POMC derivatives. This material has been shown to be composed of keratin filaments and stains with several antibodies directed against low-molecular-weight keratin proteins [275]. By electron microscopy, Crooke’s hyaline

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27

FIGURE 1.20. Corticotrophs invade the neural lobe of the pituitary (N) in a normal age-related phenomenon that is unassociated with endocrinopathy. This case, the same as that illustrated in Fig. 1.18, shows that the population of corticotrophs participating in “basophil invasion,” like those lining the pars intermedia cysts (arrow head), do not undergo Crooke’s hyaline change. (Periodic acid–Schiff stain; magnification ¥102)

FIGURE 1.19. Corticotroph with Crooke’s change in the pituitary of a patient with cortisol excess has a ring of intermediate filaments filling the cytoplasm, surrounding the nucleus and trapping numerous secretory granules in a juxtanuclear location. (Magnification ¥5090)

material is composed of an accumulation of intermediate filaments, resembling the smaller bundles seen in nontumorous corticotrophs (Fig. 1.19). The accumulation of microfilaments can be so extensive as to occupy almost the entire cytoplasm, leaving only a small juxtanuclear Golgi region and a thin rim of secretory granules adjacent to the cell membrane. Crooke’s hyaline change is reversible [195]. In the pituitaries of patients with inadequately treated adrenal insufficiency, the corticotrophs become hypertrophied, with large nuclei, prominent nucleoli, and a poorly granulated cytoplasm. In cases of long-standing Addison’s disease, focal nodular hyperplasia of corticotrophs and corticotroph adenomas may be present [277]. The ultrastructural appearance of these “adrenalectomy” cells in the human pituitary has not been well documented. However, in adrenalectomized rats, the markedly enlarged cells contain abundant rough endoplasmic reticulum and Golgi membranes and an increased number of secretory granules [278]. Chronic administration of CRH to rats increases the number of ACTH-containing cells, but no significant ultra-

structural changes have been documented [279]. Morphometric analysis indicated that there was no change in cell area; the nuclear area increased slightly and the secretory granule diameter was greater in corticotrophs of CRHexposed animals. These data suggest that the proliferation of corticotrophs in Addison’s disease may be mediated by CRH, but the morphologic characteristics of adrenalectomy cells cannot be attributed solely to CRH excess. The pars intermedia corticotrophs also are strongly (PASpositive and exhibit intense immunostaining for ACTH and other POMC derivatives [195]. The border between the pars intermedia and the pars distalis is often indistinct; occasionally a thin layer of connective tissue is noticeable between these two portions of the adenohypophysis. On the posterior aspect of the pars intermedia, basophil cells are frequently identified, often in clusters within the neuropil of the pars nervosa. These clusters vary in number and in some cases they may be found spreading deeply into the neural lobe (Fig. 1.20). This process, known as “basophil invasion,” is found in older individuals; it is more frequent and pronounced with advancing age and is said to be more prominent in men than in women. Its functional significance is not known but it is not thought to be associated with endocrine abnormalities. Some studies have suggested that the presence of a-MSH immunoreactivity is indicative of intermediate lobe differentiation [280,281]. By electron microscopy, corticotrophs of the pars intermedia are smaller, more dense, and contain fewer intermediate filaments [192]. They are also known to be less sensitive to the feedback effect of glucocorticoids, in most cases showing absent or only mild filament accumulation in patients with glucocorticoid excess and lacking the Crooke’s hyaline change that is found in pars distalis corticotrophs of those patients [195].

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The corticotrophs of the pars intermedia and those which form the basophil invasion of the pars nervosa may give rise to a specific group of corticotroph adenomas. These tumors may represent one form of the silent corticotroph adenomas which are unassociated with signs or symptoms of ACTH excess [252]; they are probably capable of secreting biologically and immunologically active substances that remain uncharacterized. As is the case with pars intermedia corticotrophs, these tumors do not show striking accumulations of intermediate filaments and, in some cases, may lack filaments totally. The derivation of corticotroph cell adenomas from this subpopulation of corticotrophs is difficult to prove. The morphology of corticotrophs in most adenomas associated with ACTH excess resembles that of nontumorous corticotrophs [195,252]. Occasional tumors are composed of cells with Crooke’s hyaline change [252], suggesting that the adenomatous cells are sensitive to feedback inhibition by glucocorticoids. Morphologic studies of functioning corticotroph adenomas in vitro [282] confirm the response of most such tumors to CRH stimulation associated with slight ultrastructural changes indicative of increased hormone synthesis. Incubation of tumor cells with glucocorticoids inhibits ACTH release and causes accumulation of intermediate filaments; however, it remains to be seen if the threshold for suppression is altered in tumor cells.

idase technique reveals granular positivity for TSH; this stain reveals the characteristic angular shape of these cells and their long cytoplasmic processes that establish contact with the basement membrane (Fig. 1.21). Ultrastructurally, these cells are characterized by their marked angularity and well-developed cytoplasmic processes [40] (Fig. 1.22). The nucleus is spherical and often eccentric while the cytoplasm contains numerous short, slightly dilated rough endoplasmic reticulum profiles, a globoid Golgi complex and small, spherical secretory granules that measure 100–200 nm in diameter. When scanty, the secretory granules characteristically are aligned under the plasmalemma and may contain rod-shaped or irregularly electron-dense contents; more densely granulated cells have granules scattered throughout the cytoplasm. Although the numbers of thyrotrophs do not appear to vary with age, these cells undergo morphologic changes in association with altered hormonal status. In patients with primary hyperthyroidism, thyrotrophs are few and small;

Thyrotrophs

The least common cell type in the adenohypophysis is the thyrotroph. These comprise approximately 5% of the total adenohypophysial cell population and are found primarily singly or in small clusters in the anteromedial portion of the gland. These medium-sized cells are basophilic when stained with conventional dyes, contain PAS positivity, stain with aldehyde fuchsin and aldehyde thionin. The immunoperox-

FIGURE 1.21. Immunohistochemical localization of thyrotropin identifies scattered angular thyrotrophs in the nontumorous human autopsy pituitary. (Magnification ¥102)

FIGURE 1.22. Nontumorous thyrotrophs have welldeveloped cytoplasm with short, slightly dilated profiles of rough endoplasmic reticulum (arrow heads), a large Golgi region (G), small secretory granules lined up along the plasma membrane and small numerous secondary lysosomes (L). (Magnification ¥7440)

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29

subset of altered somatotrophs which are bihormonal [283]; the morphologic changes are reversible in the experimental animal. Gonadotrophs

FIGURE 1.23. In a patient with long-standing hypothyroidism, the cytoplasm of these “thyroidectomy cells” is dominated by the abundant widely dilated rough endoplasmic reticulum with flocculent contents; they also contain expanded Golgi complexes (G), few small secretory granules and prominent lysosomes (L). (Magnification ¥4380)

their ultrastructural features are not well documented. In cases of untreated primary hypothyroidism, thyrotrophs are released from the negative feedback effects of thyroid hormones and TSH secretion is increased. The number and size of thyrotrophs are increased; the enlarged cytoplasm is less strongly positive using the PAS, aldehyde fuchsin and aldehyde thionin stains, and TSH immunoreactivity is faint but diffuse. These cells exhibit strong nuclear positivity for Pit-1 [195,239]. By electron microscopy, these stimulated thyrotrophs, known as “thyroidectomy cells” or “thyroid deficiency cells,” have abundant dilated rough endoplasmic reticulum cisternae and an expanded Golgi complex, whereas the number of secretory granules is reduced [40] (Fig. 1.23). Large cytoplasmic spherical, PAS-positive lysosomal globules are prominent. With prolonged stimulation, nodular hyperplasia and thyrotroph adenomas may occur [195]; TRH may be implicated in the pathogenesis of this cell proliferation [252]. In the rat, there is evidence that at least some of the thyroidectomy cells may derive from a

The cells which produce the gonadotropins, FSH and LH are the gonadotrophs; these probably represent up to 10% of the human adenohypophysial cell population [195]. No reliable cell count is, however, available. In rats, the number of gonadotrophs containing each hormone varies with age, sex, and hormonal status [40]. Gonadotrophs are scattered throughout the pars distalis and comprise the major constituent of the pars tuberalis [284]. They stain with basic dyes, the PAS technique, aldehyde thionin, and aldehyde fuchsin. The immunoperoxidase technique reveals cytoplasmic positivity for FSH and LH, often both present in the same cell, indicating that one cell type is capable of producing both gonadotropins. There are, however, variations in the number of cells containing FSH and LH, indicating that some gonadotrophs contain only one of these two hormones and there are likely fluctuations with functional demand. Nuclear reactivity for SF-1 defines the gonadotroph population [237]. The ultrastructural features of gonadotrophs reveal a single cell population which is responsible for both FSH and LH production (Fig. 1.24). The large oval or elongated cells have spherical, eccentric nuclei which are oriented with distinct polarity at the base of the cell [40,252]. Their abundant, slightly dilated rough endoplasmic reticulum profiles contain a flocculent electron-lucent substance. The Golgi is prominent and globoid, composed of sacculi and vesicles associated with immature secretory granules. Mature secretory granules are unevenly distributed in the cytoplasm and fall into two groups which vary in size and density. The distribution of these two granule populations may show sexual dimorphism. In men, smaller secretory granules are usually numerous; they have an average diameter of 250 nm. In women, larger secretory granules predominate and measure 300–600 nm in diameter. Ultrastructural immunocytology localizes FSH and LH within the same cells and, in some cases, within the same secretory granule. Gonadotrophs are found in close proximity to the basement membrane and exhibit an intriguing intimate contact with lactotrophs. The latter frequently extend cell processes around gonadotrophs and there are intercellular junctions between the two cell types [285], suggesting paracrine interactions which are not well understood. Gonadotrophs are found in the pituitaries of fetuses and show sex-related dimorphism during gestation [220]. Between 15 and 25 weeks of gestation, pituitaries of female fetuses contain more numerous gonadotrophs than pituitaries of male fetuses. Throughout gestation, LH-containing cells predominate in male pituitaries, whereas the number of LHand FSH-containing cells are almost equal in females. This dimorphism correlates with differences in the levels of hypothalamic GnRH at the same stages of gestation. In the adult

30

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Hypothalamic–Pituitary Function

FIGURE 1.24. A nontumorous gonadotroph is large and oval with short profiles of dilated rough endoplasmic reticulum containing electron-lucent contents (*), ring-like Golgi complex (G) and large, dense but irregular secretory granules. (Magnification ¥8170)

pituitary, no such dimorphism of gonadotroph numbers has been identified; however, the ultrastructural differences in males and females have been described above. Gonadotroph adenomas of the adult pituitary show an even more striking fine-structural sexual dimorphism [195,286]. Gonadotroph adenomas in women are usually well-differentiated tumors consisting of polyhedral cells with long cytoplasmic processes, dilated profiles of well-developed endoplasmic reticulum, and the characteristic “honeycomb” Golgi apparatus, a large saccular structure associated with developing secretory granules. The secretory granules in this tumor type are spherical and membrane-bound with varying electron density; they measure about 400–450 nm in diameter. The tumors in adult men are less differentiated, with poorly to moderately developed rough endoplasmic reticulum profiles, a moderately developed Golgi complex and fewer, generally smaller secretory granules. Strong nuclear SF-1 positivity is a reliable marker [237]. Gonadotrophs of the pars distalis show morphologic changes which reflect their hormonal environment; in con-

trast, the gonadotrophs of the pars tuberalis show signs of functional inactivity in most circumstances. In patients treated with pharmacologic doses of estrogen, the gonadotroph cells are small and dense [195]. During pregnancy, the number of cells immunoreactive for FSH and/or LH is also significantly reduced [264]. Gonadotrophs are morphologically abnormal in Kallmann’s syndrome, an uncommon variant of hypothalamic hypogonadism due to GnRH deficiency and associated with anosmia [182]; there is a marked decrease in the number of these cells and the remaining small cells are only weakly positive for FSH and LH. Castration leads to characteristic morphology in gonadotrophs [40,195,287]. Prolonged lack of the negative feedback effect of gonadal steroids results in stimulation of gonadotrophs to secrete FSH and LH in higher quantities. These stimulated cells are enlarged and are found in increased numbers. They have vacuolated cytoplasm which may take the form of several small vacuoles or one large vacuole; the latter displaces the nucleus to the cell periphery, giving the cell a “signet ring” appearance. By electron microscopy, these so-called “gonadectomy cells,” “gonadal deficiency cells,” or “castration cells” (Fig. 1.25) have markedly dilated rough endoplasmic reticulum, which is responsible for the cytoplasmic vacuoles seen by light microscopy. The Golgi complex is enlarged and hormone storage is decreased but not lost, despite the marked increase in secretory activity, since secretory granules are present in reduced numbers. It has been suggested that secretory granule formation is bypassed and that discharge of hormone takes place in an unconventional way not discernible by electron microscopy. An unusual prominence of large active gonadotrophs has been noted in the nontumorous adenohypophysis of some women with PRL-producing adenomas [40]. The factors underlying this change are not clear, but it has been suggested as the explanation for the LH hyperresponse to GnRH which has been found in these patients. Follicular Cells

Follicular cells are found throughout the adenohypophysis surrounding follicles, lumina lined mainly by agranular or poorly granulated cells that are joined at their apex by junctional complexes [195,288]. By electron microscopy, it was shown that granulated adenohypophysial cells can form follicles around damaged cells which have disrupted cell membranes. The surrounding cells form specialized intercellular attachments and the participating cells undergo degranulation and dedifferentiation. These follicular cells may derive from somatotrophs, lactotrophs, or corticotrophs. The follicular cells form specialized junctions, macula adhaerens, between each other and with adjacent granulated adenohypophysial cells; in contrast, granulated adenohypophysial cells form only the less prominent zonulae adhaerentes. Follicles are found in areas with increased cell destruction, particu-

Chapter 1

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31

FIGURE 1.26. Immunolocalization of S-100 protein identifies a pituitary stellate cell with long branched cytoplasmic processes surrounding granulated adenohypophysial cells. (Magnification ¥256)

FIGURE 1.25. After castration, “gonadectomy cells” have markedly dilated rough endoplasmic reticulum and large globoid Golgi regions (G) which harbor forming secretory granules. (Magnification ¥4620)

been implicated as the source of FGF and vascular endothelial growth factor [293], but are not found in the pituitaries of anencephalics [294]. They are numerous in the compressed adenohypophysis at the periphery of adenomas. Some investigators have found no S-100-reactive sustentacular cells within pituitary adenomas [290]; in contrast, others have identified S-100-containing cells within tumors of several types [295]. They are also found in large numbers at the periphery of other pituitary lesions, such as abscesses, amyloid deposits and in the residual hypophysis after surgery, but not adjacent to metastatic tumor deposits, infarcts, or Rathke’s cleft cysts [296]. Null Cells

larly surrounding tumors [40]. They are assumed to play a role in isolating and processing cell debris. Some pituitary adenomas contain follicles. Folliculostellate Cells

Immunocytochemical studies have localized S-100 protein to a specific subtype of cells in the normal human pituitary gland [289,290]. These cells are agranular and do not contain immunoreactive hormones. Some also contain immunoreactivity for glial fibrillary acidic protein (GFAP). They have a characteristic morphology, with long, branched cytoplasmic processes embracing granulated adenohypophysial cells (Fig. 1.26). Because of confusion with the follicular cells described above, some authors have suggested that these cells be called “stellate cells” [289]. They are believed to have a supportive role similar to that played by the S-100-positive sustentacular cells of the adrenal medulla and carotid body. In addition, they are thought to play a role in paracrine regulation [291] and have been shown to produce IL-6, a cytokine which may participate in local regulation of hormone secretion [292]. These cells have also

The term null cell was used to identify cells that show evidence of adenohypophysial differentiation, possessing organelles required for hormone synthesis and storage, but whose differentiation is incomplete and does not resemble any of the known adenohypophysial cell types [297]. The term was applied initially to a group of tumors which possessed no histologic, immunocytochemical, or ultrastructural markers revealing their cellular differentiation and which were unassociated with clinical evidence of hormone excess. Application of increasingly sensitive techniques of cell identification have shown that most tumors with morphologic features of null cells are in fact gonadotroph adenomas [298,237]; however, only a small subpopulation of tumor cells may be hormonally active at any one time, as indicated by the reverse hemolytic plaque assay [299]. Immunohistochemical analyses [221] and studies of hormone mRNA [300] have shown that the majority of these tumors contain cells capable of producing glycoprotein hormone subunits. Focal ultrastructural differentiation along several lines has also been documented [301]. It has been suggested that these tumors may originate from uncommitted or

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committed stem cells or from dedifferentiated adenohypophysial cells. Null cells may also represent unstimulated or resting cells that are not actively engaged in hormone production and cannot be recognized with currently available investigative techniques [302]. The nontumorous pituitary contains cells that cannot be identified conclusively by ultrastructural criteria. These may represent resting cells, uncommitted or committed stem cells. In the fetal pituitary, a population of cells with features of the glycoprotein hormone cell line can be identified prior to the recognition of differentiated thyrotrophs or gonadotrophs [221]; a-subunit immunoreactivity is present in fetal glands at the same stage of gestation and b-subunits are not yet detected [220]. It has been suggested that these primitive cells which resemble null cells may represent the source of a-subunit and may be precursors of the glycoprotein hormone cell line; they may be the cell of origin of some null cell adenomas. The null cells are chromophobic and by immunocytochemistry, usually contain no hormones [195,252]. A small number of these cells may contain various pituitary hormones, most commonly a-subunit of the glycoprotein hormones followed by b-subunits of FSH and/or LH. They usually exhibit SF-1 nuclear staining, indicating gonadotrophin differentiation [237]. By electron microscopy, the cells are polyhedral with irregular nuclei. The poorly developed cytoplasm contains short, scattered rough endoplasmic reticulum profiles, an inconspicuous or only moderately developed Golgi apparatus and small, dense, rodshaped mitochondria. Secretory granules are few and small, measuring less than 250 nm in diameter, frequently with central electron-dense cores, peripheral electron-lucent halos, and a prominent limiting membrane (Fig. 1.27). Oncocytes

Oncocytes are large cells which have abundant acidophilic granular cytoplasm due to the numerous mitochondria that take up acid dyes [195,252]. By electron microscopy, they are characterized by a striking increase in the number and size of mitochondria. A varying number of oncocytic cells may be found in the normal human hypophysis [303]. The number of these cells appears to increase with advancing age, a phenomenon also found in other organs including thyroid, parathyroid, and salivary glands. Cells in hormonally active adenomas only rarely show oncocytic transformation; the tumors usually retain their fine structural and immunocytochemical markers and can be recognized as showing various degrees of oncocytic change. In contrast, null cells, which have no other identifying markers, more frequently undergo oncocytic change. Gonadotroph adenomas regularly contain a varying number of cells with oncocytic change, and the number of oncocytes in any given tumor often increases with tumor recurrence [195]. Oncocytomas are found more commonly in elderly patients. In vitro studies using tissue culture techniques [298] and the reverse hemolytic plaque

FIGURE 1.27. A null cell in the nontumorous human pituitary has poorly developed organelles in a scant cytoplasm. However, there are short profiles of rough endoplasmic reticulum (arrow heads) and a few small secretory granules, indicating the potential for hormone synthesis. This cell has no features which allow recognition of its differentiation. (Magnification ¥5950)

assay [299] have indicated that there is no functional difference between null cell adenomas and oncocytomas of the human pituitary. The factors underlying oncocytic change are unknown, but they may lie in abnormalities of the mitochondrial DNA which is independent of the cellular DNA [304]. Oncocytes are large polyhedral cells with abundant granular cytoplasm that varies from strongly acidophilic to chromophobic. These cells may show ill-defined purple coloration with trichrome stains using aniline blue; this may lead to misinterpretation as basophilia. The phosphotungstic acid–hematoxylin method is helpful to demonstrate mitochrondrial abundance. Oncocytes showing ultrastructural features of differentiated adenohypophysial cells may contain the appropriate immunoreactive hormone. The majority of oncocytes, which are thought to be derived from null cells of gonadotrophs contain no immunoreactive hormones or may contain a-subunit, FSH LH. They often have nuclear SF-1 reactivity [237].

Chapter 1

FIGURE 1.28. In the nontumorous human pituitary, an isolated oncocyte has abundant cytoplasm which is almost totally occupied by numerous spherulated mitochondria. Rough endoplasmic reticulum (arrow heads), a small Golgi complex (G) and small secretory granules suggest that the cell is capable of hormone synthesis and storage. (Magnification ¥5590)

By electron microscopy, oncocytes are readily identified as large, polyhedral cells with irregular nuclei and a cytoplasm almost totally filled with numerous mitochondria. Histologically some oncocytes are dark or acidophilic; these cells contain tightly packed oblong mitochondria with granular matrix and numerous transverse cristae. Other oncocytes may be histologically light, i.e., only slightly acidophilic or chromophobic; their crowded mitochondria appear swollen and rarefied, with loss of cristae. Oncocytes contain small, short profiles of rough endoplasmic reticulum, inconspicuous Golgi regions and few scattered secretory granules (Fig. 1.28). Others

Acidophil Stem Cells are recognized primarily in pituitary adenomas producing both GH and PRL [195,305]. They are rare and are not known to occur in the nontumorous pituitary. The cell type is assumed to derive from a common precursor of somatotrophs and lactotrophs; while

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mammosomatotrophs have been recognized as the source of GH and PRL early in gestation, no cells resembling tumorous acidophil stem cells have yet been identified in the human fetal pituitary [221]. Immunohistochemistry reveals the presence of PRL and GH in the cells. The positivity for PRL is usually stronger and patients with tumors composed of these cells have hyperprolactinemia; GH production may be reflected in the phenomenon known as “fugitive acromegaly.” By electron microscopy, these cells resemble sparsely granulated lactotrophs and have some features of somatotrophs. They are irregular in shape and have large nuclei containing fine chromatin and inconspicuous nucleoli. The cytoplasm contains scattered rough endoplasmic reticulum profiles and relatively small Golgi regions. The Golgi sacculi are flat and may contain a few forming granules. The mitochondria may be abundant, indicating oncocytic transformation, and often show focal cavitation as well as a unique mitochondrial gigantism not identified in other pituitary cell types. Giant mitochondria may reach the size of a nucleus and be visible by light microscopy as cytoplasmic vacuoles. The mitochondria retain their double limiting membranes but lose their cristae and are filled with electron-lucent granular matrix. Occasionally, they contain electron-dense tubular structures resembling tubules of centrioles and cilia. The secretory granules in this cell type are sparse and small, ranging from 50 to 300 nm. Characteristically, granule extrusions or “misplaced exocytoses” are identified, similar to those of sparsely granulated lactotrophs. In addition, these cells have a structural marker of sparsely granulated somatotrophs, the fibrous body, a juxtanuclear aggregate of smooth endoplasmic reticulum membranes, and keratin immunoreactive intermediate filaments. These features of lactotrophs and somatotrophs are frequently found in the same cell; ultrastructural immunocytochemistry confirms the localization of GH and prolactin in the same cell. A tumor type identified as silent subtype III adenoma is composed of chromophobic or slightly acidophilic cells which contain immunocytochemical evidence of multiple hormones. Staining for ACTH is generally weak, bendorphin immunoreactivity may be strong and the tumor cells may contain GH, PRL, and/or a-subunit immunoreactivity. In some cases, the tumors contain no recognizable hormones. By electron microscopy, large, elongated cells have an eccentric nucleus, a prominent nucleolus which may harbor spheridia, and a highly differentiated cytoplasm with abundant rough and smooth endoplasmic reticulum profiles and a well-developed Golgi apparatus. The secretory granules are small, measuring less than 200 nm in diameter, and have variable electron density; they are mainly spherical but occasionally irregular or dropshaped granules are found. These cells bear no similarity to any of the known nontumorous adenohypophysial cells and their cytodifferentiation is unknown. Other unclassified plurihormonal cells have been identified in the nontumorous pituitary and adenomas [40,195]. The

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most common types produce GH, a-subunit, TSH, and/or PRL in any combination. Some of these cells have ultrastructural features of somatotrophs or mammosomatotrophs, while others resemble thyrotrophs [40]. Studies of the rat pituitary have confirmed the existence of these cells and indicate a role for them in some pathologic states; for example, some somatotrophs transform into a population of thyroidectomy cells in hypothyroidism [283]. This common pattern of plurihormonality is easily explained by the expression of Pit-1, the transcription factor implicated in activation of the genes encoding GH, PRL and bTSH. However, occasionally other patterns of plurihormonality have been reported. Very rarely, cells producing POMC derivatives with ultrastructural features of corticotrophs may produce gonadotropins or other hormones both in human tumors [195,306] and in the rat nontumorous pituitary [307]. Conversely, cells with gonadotroph morphology may contain ACTH as well as gonadotropins [308]; it is intriguing, however, that ACTH, which is so commonly expressed in extrapituitary tumors, is rarely found in plurihormonal cells of the adenohypophysis.

Localization of Other Peptides in the Adenohypophysis A number of substances not initially thought to be of pituitary origin are detectable in adenohypophysial cells. Hypothalamic Adenohypophysiotropic Hormones

GHRH, somatostatin, TRH, and GnRH have been identified in pituitary adenohypophysial cells [308–311]. Large amounts of GHRH and somatostatin were measured in culture media of perifused normal pituitaries and GHsecreting adenomas and their release was stimulated by other peptides [309]. Preprosomatostatin mRNA has been detected in nontumorous adenohypophysis as well as several types of pituitary adenomas [310]. It is known that GnRH binds to gonadotrophs and becomes internalized [311]; however, it has also been shown that TRH and GnRH are released by pituitary cells maintained in culture for a prolonged period [308]. GnRH is colocalized with b-LH and some gonadotrophs also contain TRH. These data suggest production of hypothalamic peptides by de novo synthesis in a multipotential pituitary cell. Angiotensin II

Angiotensin II, as well as renin and angiotensin converting enzyme, have been localized in pituitary gonadotrophs [312]. These substances colocalize with b-LH. Angiotensin II has been shown to stimulate PRL release in vitro; this is a dosedependent phenomenon. It has also been found to stimulate ACTH and b-endorphin release. Lactotrophs and corticotrophs have high-affinity binding sites for angiotensin II. Bombesin/GRP

Bombesin-like immunoreactivity has been found in the pituitary gland [313]; however, cell localization has not been

demonstrated. Bombesin-related peptides stimulate pituitary hormone release, mediated by the hypothalamus. CGRP

CGRP is found in the pituitary in small amounts and the concentration is known to increase with age. It is unclear if this substance is present in adenohypophysial cells or in nerve terminals of the posterior lobe [314], since CGRP is found in the hypothalamus (see above). CCK

CCK is found in the adenohypophysis but in a form which differs from that present in other CCK-synthesizing tissues [315]. The pituitary is devoid of CCK-8 but contains substantial amounts of three large nonamidated pro-CCK fragments and small amounts of a-amidated CCK (CCK-58, CCK-33, component I). No immunocytochemical localization has been reported but CCK has been found in corticotroph adenomas in large amounts and in some tumors associated with acromegaly which also contained ACTH. It therefore seems that pituitary CCK derives from corticotrophs [316]. The form found in tumors is smaller and less sulfated than in the normal pituitary; tumors contain CCK-8-like forms similar to those found in brain and gut [315]. Galanin

In humans, galanin has been localized in corticotrophs of the nontumorous pituitary, including Crooke’s cells and cells of basophil invasion in the posterior lobe. Galanin is also present in some corticotroph adenomas [139,317]. In contrast, in rats, galanin mRNA is found in estrogen-induced pituitary tumors [318] and galanin has been localized in PRL- and GH-producing cells as well as some TSHcontaining cells [319]. The reason for this species-specific regulation is unknown. In the rat, galanin expression shows sexual dimorphism [319], is inducible by estrogen [318–320] and is altered by changes in adrenal, thyroid, or gonadal status [319]. It appears to be involved in the modulation of pituitary GH and PRL secretion, probably mediated by alterations in their hypothalamic regulation [140,141]. Gastrin

The neurohypophysis is known to contain gastrin [142], but there appears to be species-specific variation in the adenohypophysis. The human adenohypophysis contains only traces of this substance which is localized to corticotrophs [321] and corticotroph adenomas [322]. Analogy has been drawn to cosynthesis with POMC in other tissues; the significance of this colocalization is unknown and gastrin is not thought to act directly on the pituitary but rather in the hypothalamus. NPY

NPY immunoreactivity and mRNA are found in the pituitary and can be localized by immunocytochemistry

Chapter 1

in adenohypophysial cells consistent with a subset of thyrotrophs [323]. These cells increase in size and NPYimmunoreactivity after thyroidectomy. NPY may have autocrine or paracrine activity in the modulation of release of GH and gonadotropins. Neurotensin

Neurotensin has been localized to the anterior pituitary and is thought to be present in gonadotrophs. It is also known to be affected by thyroid status, being found in reduced amounts in both hypothyroidism and hyperthyroidism [151,324]. The role of neurotensin in the pituitary is not known. Substance P

Immunoreactive substance P has been found in the rodent anterior pituitary. In rats it is localized in gonadotrophs and lactotrophs [325], but in the guinea pig it is found in thyrotrophs [326]. Thyroidectomy increases and thyroid hormone treatment decreases the amount of substance P in the pituitary [152]; the amount also varies with levels of gonadal steriods. It is not clear if thyroid hormone and corticosteroids act directly to influence pituitary content. Substance P may play a local or paracrine role in the adenohypophysis. VIP

There is evidence that VIP is synthesized in the pituitary [327] in lactotrophs [127,328]; it is also inducible by estrogen and may be altered by other changes in the hormonal environment. Recently, it was found to be present in increased amounts in hypothyroidism [329] and VIP production by adenohypophysial cells in vitro was found to be stimulated by TRH as well as GHRH, but not by CRH or GnRH. Tissue culture studies have shown that VIP regulates tonic PRL secretion but does not affect TRH- or GHRHinduced PRL release [329]. Cytokines

Cytokines are known to modulate pituitary function and some have been shown to be produced in the hypothalamus (see above and references 161–166). IL-6 has been demonstrated in stellate cells within the pituitary [292] and is known to modulate GH and PRL responses to appropriate stimuli [164]. Growth Factors

Growth factors regulate cell replication and functional differentiation by directly altering the expression of specific genes. Several polypeptide growth factors regulate anterior pituitary hormone secretion. Growth factor action in the pituitary, however, must be understood in the context that the pituitary itself is a site of both synthesis and action of growth factors [330,331]. Several pituitary-derived growth factors are also regulated within the pituitary by peripheral hormones including triiodothyronine and hydrocortisone.

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Pituitary-derived growth factors include EGF, FGF, TGF-a and TGF-b, IGF, endothelin, and IL-1 [331]. Several partially characterized growth factors have also been described, including thyroid hormone-inducible growth factor, vascular endothelial growth factor mammary cell growth factor, chondrocyte growth factor, mammary cell growth factor, chondrocyte growth factor, and adipocyte growth factor [331]. EGF, a potent stimulator of PRL and ACTH secretion in vitro, also attenuates GH secretion [332–334]. EGF may also play a role in the regulation of LH secretion [335]. EGF has been localized in the pituitary within cells that produce glycoprotein hormones. EGF-receptor is expressed by normal human anterior pituitary and is upregulated in some adenomas, usually recurrent somatotroph tumors [336]. It has been speculated that EGF may mediate proliferation of thyrotrophs in hypothyroidism and of lactotrophs in the lactotroph hyperplasia of pregnancy [337]. TGF-a is expressed by pituitary cells where it has a membrane-anchored localization [338,339]; by immunohistochemistry it colocalizes with PRL and possibly with GH in the bovine pituitary [340]. TGF-a interacts with the EGF receptor and inhibits replication of rat pituitary tumor cells [341]. The regulation of lactotroph growth and differentiation by this substance suggests a potential role in promotion or facilitation of pituitary tumorigenesis [339] and in transgenic mice, targeting of TGF-a overexpression to lactotrophs results in lactotroph adenoma formation [343]. Activin, a member of the TGF-b gene family, suppresses GH secretion and somatotroph replication, and stimulates FSH secretion [343–346]. The inhibin subunits which comprise activin and inhibin and their mRNAs have been localized in pituitary gonadotrophs [309]. Activin-binding protein, which shows homology to follistatin, has been isolated in the rat pituitary and appears to block the action of activin [347]. Follistatin is expressed by normal gonadotrophs and is reduced or absent in gonadotroph adenomas, suggesting a role for this growth factor in the tumorigenic process [348]. The pituitary contains high concentrations of basic FGF [349]. There are several indications that the FGF family plays an important role in the regulation of pituitary function. It may be an important paracrine regulator of PRL secretion [170]; nonmitogenic concentrations of FGF stimulate TRHinduced PRL and TSH secretion in pituitary cells. Pituitaryderived FGF stimulates the replication of human pituitary cells in vitro [350]. FGF has also been implicated in the new arteriolar vessel formation reported to accompany prolactinomas [351]. FGF expression is upregulated in aggressive pituitary adenomas and this growth factor has been shown to be released by the adenoma cells [352], indicating its potential role in the control of adenohypophysial cell proliferation. Moreover, altered FGF receptors have been implicated in the dysregulated growth of pituitary tumors [353].

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Endothelin, a powerful vasoconstrictor, is expressed in the pituitary and stimulates FSH and LH secretion [354]. Pituitary-derived endothelin-3 is stimulated by IGF-I [355]. IGF-I mediates most of the peripheral growth promoting actions of GH [356]. GH-dependent IGF-I is secreted predominantly by the liver, and IGF-I participates in feedback regulation of GH gene transcription and secretion [357]. Regulation of pituitary IGF-I receptors also determines the action of the growth factor on somatotroph function [356]. IGF-I also stimulates the in vitro secretion of hypothalamic SRIH, further delineating its role in feedback regulation of GH secretion [358]. Pituitary expression of GH, IGF-I and IGF-I receptors have been documented [359,360] and may therefore mutually interact by a paracrine or autocrine feedback mechanism [361]. Regulation of endogenous or circulating pituitary growth factors and their respective receptors may be important paracrine or autocrine determinants of pituitary cell function and trophic hormone secretion. The role of these growth factors in the development of pituitary tumors is being intensively studied [232].

Classification of Adenohypophysial Pathology Many diseases can affect tissues in and adjacent to the sella turcica, giving rise to a wide spectrum of clinical pathology. The clinical problems may be manifest as pituitary hormone deficiency or pituitary hormone excess. In addition, associated problems arise from compression, invasion, injury, or destruction of other tissue components; symptoms such as visual field defects, nerve palsies and headaches are characteristic of mass effects on the hypothalamus and neurohypophysis. Adenohypophysial Hormone Deficiency

The clinical problem of pituitary hormone deficiency may be due to decreased secretion or to increased degradation of hormone. Characteristic adenohypophysial changes are often associated with abnormalities of hormone secretion. Hypopituitarism involving one, two, or more adenohypophysial hormones is usually attributed to either a decreased number of hormone-secreting cells or to reduced endocrine activity. Organic lesions such as massive necrosis, inflammatory, or neoplastic processes can replace large portions of the pituitary and account for reduction of hormone-secreting parenchyma [195]. Reduced endocrine activity can result from target gland hormone excess or administration of exogenous hormones feedback negatively to suppress hypophysial function. Alternatively, it may be due to decreased hypothalamic stimulation or interference with receptors which govern secretion of hormones. Various developmental abnormalities can reduce the amount of adenohypophysial parenchyma. Some of these have been reviewed above (see embryology). It is now recognized that mutations of PROP-1 or Pit-1 result in various patterns of pituitary hormone deficiency and hypoplasia

[229,230,233,245,247]. In pituitary aplasia, there is total absence of the adenohypophysis due to a defect in the formation of Rathke’s pouch; pituitary hypoplasia is a milder variant of this disorder. In anencephaly, the brain, including the hypophysiotropic hypothalamus, is missing; due to lack of neurohumoral control, the adenohypophysis is smaller than normal and there is marked reduction in the number of corticotrophs associated with a less striking reduction of other adenohypophysial cell types [276a]. Cornelia de Lange syndrome, a rare congenital cyst of Rathke’s pouch, may compress the adenohypophysis and lead to various degrees of hypopituitarism. Pituitary dystopia or ectopia rarely affects function. Tissue destruction leads to a decrease or absence of pituitary hormone-producing parenchyma. This may be caused by tumors, inflammatory or infiltrative processes, or vascular lesions. Symptoms of hypopituitarism develop only with reduction of more than 60% of anterior lobe tissue and is clinically significant when there is more than a 90% reduction of hormone-producing cells. Hormonally inactive tumors of the adenohypophysis represent approximately 25% of surgically removed pituitary adenomas [195]. The null cell adenomas and oncocytomas fall into this category; these cell types have been discussed above. Craniopharyngiomas are less common than pituitary adenomas [195]. They represent 3–5% of intracranial neoplasms and occur usually in children or young adults. They are more frequently suprasellar than intrasellar and can cause varying degrees of hypopituitarism due to compression or destruction of the adenohypophysis, pituitary stalk, or hypophysiotropic hypothalamus. Other primary neoplasms in and around the sella turcica include fibromas, angiomas, gliomas, meningiomas, granular cell tumors, paragangliomas, chordomas, and many others (see Chapter 15) [195]. These tumors produce no hormones. The majority are benign and may be discovered incidentally at postmortem examination. Occasionally they cause hypopituitarism and local symptoms. Primary sarcomas have been reported in patients who have previously received irradiation for the treatment of pituitary adenomas [363]. Metastatic malignancies are found in patients with widely disseminated neoplasms; reported incidences vary from 1 to 14% [47,362]; however, the true incidence of adenohypophysial involvement is not known, since small deposits are easily overlooked. Pituitary involvement is rarely manifest before patients are overwhelmed by the malignancy. Inflammatory conditions in the sella turcica may occur as acute processes, with purulent hypophysitis and abscess formation, due to severe septicemia or direct extension from adjacent tissues. Granulomatous inflammation may be seen in tuberculosis, syphilis, sarcoidosis, and giant-cell granuloma. The latter is an uncommon disorder of unknown etiology which may involve the pituitary gland selectively; it may represent localized sarcoidosis [364]. Lymphocytic hypophysitis is an autoimmune disorder which may cause

Chapter 1

extensive destruction of the adenohypophysis and subsequent hypopituitarism [365,366]. Vascular lesions such as hemorrhages may occur as complications of traumatic head injuries or in tumors. Infarction is seen focally as an incidental autopsy finding but significant lesions may be associated with diabetes mellitus, generalized carcinomas, cranio-cerebral trauma, increased intracranial pressure, infection, and in patients maintained on mechanical ventilators. Postpartum pituitary necrosis, known as Sheehan’s syndrome [367], occurs in pregnancy, during which time the pituitary is predisposed to vasospasm and infarction. When damage is significant, hypopituitarism is proportional to the degree of tissue destruction. Infiltrative disorders such as amyloidosis, hemochromatosis, and mucopolysaccharidosis, may impair adenohypophysial cell function. Hemochromatosis can give rise to selective hypogonadism due to preferential deposition of iron in pituitary gonadotrophs [368]. Feedback inhibition may cause cytologic alterations in the adenohypophysis. These changes have been described above. Selective deficiencies of adenohypophysial hormones may result from inherited genetic abnormalities or selective damage to one cell type. Isolated GH deficiencies are a genetically heterogeneous group of disorders that cause dwarfism. In most cases, there is a normal pituitary with normal numbers of granulated somatotrophs; these are attributed to GHRH deficiency or abnormality [172–175]. Selective deficiency of a specific pituitary transcription factor, Pit-1, causes selective congenital GH and PRL deficiency associated with TSH deficiency [369–371]; several animal models of pituitary dwarfism, the Snell, Jackson and Ames dwarf mice, have also been shown to have specific abnormalities of the Pit-1 gene [372]. Congenital isolated ACTH deficiency has been attributed to T pit mutations. Lymphocytic hypophysitis has been reported to cause selective destruction of corticotrophs; antibodies to other pituitary cell types are also detectable in patients with endocrine autoimmune diseases and may give rise to isolated pituitary hormone deficiency [365,366]. Short stature with features of isolated GH deficiency but elevated GH blood levels was found to be due to a deficiency of GH-binding protein, the extracellular domain of the GH receptor [373,374]. Adenohypophysial Hormone Excess

Excess hormone production by the pituitary is usually due to a pituitary tumor [195]. The most common lesion is adenoma; carcinoma occurs exceedingly rarely and may be associated with hormone excess. Hyperplasia and hypertrophy of hormone-secreting cells may result from increased stimulation by hypothalamic releasing hormones, or from loss of inhibition by hypothalamic-inhibiting hormones, or excess target organ hormones. The cytologic features of each adenoma type have been described above. The features of hyperplastic adenohypophysial cells have also been provided. Tumor-like lesions may also be associated with hypersecretion of adenohypophysial hormones. Lymphocytic

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hypophysitis may give rise to a mass lesion of the sella turcica associated with hyperprolactinemia [365,366]. The elevated PRL levels may be due to inflammation, injury of the stalk or hypothalamus or compression of those structures by the enlarged, inflamed gland. The disorder is frequently associated with pregnancy and it has been suggested that antibodies directed against hyperplastic lactotrophs may stimulate PRL release, interfere with dopamine receptors, or damage PRL cells causing release of stored PRL. Rarely, pituitary hormone excess has been associated with normal pituitary morphology [195]. The hyperfunction may be caused by intrinsic abnormalities of the cells which produce subtle changes that may not be detectable by conventional morphologic techniques.

REFERENCES 1 Descartes R. Des Passions de l’Ame. Amsterdam, 1649. 2 Zander R. Ueber functionelle und genetische Beziehungen der Nebennieren zu anderen Organen, speciell zum Grosshirn. Beitr Pathol Anat 1890;7: 439–534. 3 Bernard C. Physiologie; chiens rendus diabétiques. C R Soc Biol 1849;1:60. 4 Marie P. Sur deux cas d’acromégalie. Hypertrophie singuliére non congénitale des extremités supérieures, inférieures et céphaliques. Rev Méd 1886;6:297–333. 5 Minkowski O. Ueber einen Fall von Akromegalie. Berl Klin Wochenschr 1887;21:371–374. 6 Cushing H. The Pituitary Body and Its Disorders. Philadelphia: JB Lippincott, 1912. 7 Simmonds M. Zwergwuchs bei Atrophie der Hypohysen Vorderlappens. Munch Med Wochenscht 1919;45:487–488. 8 Harris GW. Neural control of pituitary gland. Physiol Rev 1948;28: 139–179. 9 Du Bois Reymond E. Gesammelte Abhandlungen zur allgemeinen Muskel und Nerven physic, vol. 2. Leipzig: Veit & Comp, 1877. 10 Schiefferdecker. Ueber die Neuronen und die innere Sekretion. Bonn: Sitzgsber Niederrhein Ges Natur Heilkde, 1905:46–54. 11 Lieutaud J. Essais Anatomiques, contenant l’histoire exact de toutes les parties qui composent le corps de l’homme, avec la manière de dissequer. Paris: Huart, 1742. 12 von Luschka H. Der Hirnanhang und die Steissdruese des Menschen. Berlin: G Reimer, 1860:1–97. 13 Popa GT, Fielding U. The vascular link between the pituitary and the hypothalamus. Lancet 1930;ii:238–240. 14 Scharrer E, Scharrer B. Secretory cells within the hypothalamus. Res Publ Assoc Nerv Ment Dis 1940;20:170–174. 15 Bargmann W, Scharrer E. The site of origin of the hormones of the posterior pituitary. Am Sci 1951;39:255–259. 16 du Vigneaud V, Gish DT, Katsoyannis PG. A synthetic preparation possessing biological properties associated with arginin-vasopressin. J Am Chem Soc 1954;76:4751–4752. 17 Guillemin R. Peptides in the brain: the new endocrinology of the neuron. Science 1978;202:390–402. 18 Bøler J, Enzman F, Folkers K et al. The identity of chemical and hormonal properties of the thyrotropin-releasing hormone and pyroglutamylhistidyl proline amide. Biochem Biophys Res Commun 1969;37:705–710. 19 Burgus R, Dunn TF, Desiderio D et al. Structure moléculaire du facteur hypothalamique TRH d’origine ovine: mise en évidence par spectrométrie de masse. C R Acad Sci 1969;269:1870–1873. 20 Baba Y, Matsuo H, Schally AV. Structure of the porcine LH- and FSHreleasing hormone. II. Confirmation of the proposed structure by conventional sequential analysis. Biochem Biophys Res Commun 1971;44:459–467. 21 Burgus R, Butcher M, Ling N et al. Structure moléculaire du facteur hypothalamique (LRF) d’origine ovine contrôlant la sécrétion de l’hormone gonadotrope hypophysaire de lutéinisation. C R Acad Sci 1971;173: 1611–1613. 22 Schally AV, Arimura A, Baba Y et al. Isolation and properties of the FSH and LH releasing hormone. Biochem Biophys Res Commun 1971;43:393–399.

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Hypothalamic–Pituitary Function

23 Brazeau P, Vale W, Burgus R et al. Hypothalamic polypeptide that inhibits the secretion of immunoreactive pituitary growth hormone. Science 1973;179: 77–79. 24 Vale W, Spiess J, Rivier C et al. Characterization of a 41-residue ovine hypothalamic peptide that stimulates secretion of corticotropin and aendorphin. Science 1981;213:1394–1397. 25 Guillemin R, Brazeau P, Böhlen P et al. Growth hormone-releasing factor from a human pancreatic tumor that caused acromegaly. Science 1982;218: 585–587. 26 Rivier J, Spiess J, Thorner M et al. Characterization of a growth hormone releasing factor from a human pancreatic islet tumour. Nature 1982;300: 276–277. 27 Benda C. Beitrage zur normalen und Pathologischen Histologie der menschlichen Hypophysis cereberi. Berl Klin Wochenschr 1900;37:1205–1210. 28 Erdheim J. Zur normalen und pathologischen Histologie der Glandula Thyreoidea, Parathyreoidea und Hypophysis. Beitr Pathol Anat Allg Pathol 1903;33:158–236. 29 Kraus EJ. Zur Pathologie der basophilen Zellen der Hypophyse. Zugleich ein Beitrag zur Pathologie des Morbus Basedowi und Addisoni. Virchows Archiv 1923;247:421–447. 30 Severinghaus AE. Cellular changes in the anterior hypophysis with special reference to its secretory activities. Physiol Rev 1937;17:556–588. 31 Le Gros Clark WE. Morphological aspects of the hypothalamus. In: Le Gros Clark WE, Beattie J, Riddoch G, Dott NM, eds. The Hypothalamus: Morphological, Functional, Clinical and Surgical Aspects. Edinburgh: Oliver & Boyd, 1938:1–68. 32 Rasmussen AT. The proportions of various subdivisions of the normal adult human hypophysis cerebri and the relative number of the different types of cells in pars distalis, with biometric evaluation of age and sex differences and special consideration of basophilic invasion into the infundibular process. Res Publ Assoc Res Nerv Ment Dis 1938;17:118–150. 33 Romeis B. Hypophyse. In: von Mollendorf W, ed. Handbuch der Mikroskopischen Anatomie des Menschen, vol. 6, part 3. Berlin: Springer-Verlag, 1940:1–625. 34 Phifer RF, Spicer SS, Hennigar GR. Histochemical reactivity and staining properties of functionally defined cell types in the human adenohypophysis. Am J Pathol 1973;73:569–588. 35 Herlant M. Étude critique de deux techniques nouvelles destinées à mettre en évidence les différentes catégories cellulaires présentes dans la glande pituitaire. Bull Micro Appl 1960;10:37–40. 36 Brookes LD. A stain for differentiating two types of acidophil cells in the rat pituitary. Stain Technol 1968;43:41–42. 37 Sternberger LA. Immunocytochemistry. Englewood Cliffs: Prentice-Hall, 1974. 38 Kovacs K, Horvath E, Ryan N. Immunocytology of the human pituitary. In: DeLellis RA, ed. Diagnostic Immunohistochemistry. New York: Masson Publishing Inc, 1981:17–35. 39 Rinehart JF, Farquhar MG. Electron microscopic studies of the anterior pituitary gland. J Histochem Cytochem 1953;1:93–108. 40 Horvath E, Kovacs K. Fine structural cytology of the adenohypophysis in rat and man. J Electr Microsc Tech 1988;8:401–432. 41 Moriarty GC. Adenohypophysis: ultrastructural cytochemistry. A review. J Histochem Cytochem 1973;21:855–892. 42 Bendayan M. Protein A—gold electron microscopic immunocytochemistry: methods, applications and limitations. J Electron Microsc Tech 1984;1:243–270. 43 Neill JD, Frawley LS. Detection of hormone release from individual cells in mixed populations using a reverse hemolytic plaque assay. Endocrinology 1983;112:1135–1137. 44 Asa SL. In vitro culture techniques. In: Kovacs K, Aas SL, eds. Functional Endocrine Pathology, 2nd ed. Boston: Blackwell Science, 1998:118–133. 45 Lloyd RV. Use of molecular probes in the study of endocrine diseases. Human Pathol 1987;18:1199–1211. 46 Sheehan HL, Kovacs K. Neurohypophysis and hypothalamus. In: Bloodworth JMB Jr, ed. Endocrine Pathology, General and Surgical, Baltimore: Williams & Wilkins, 1982:45–99. 47 Scheithauer BW. The hypothalamus and neurohypophysis. In: Kovacs K, Asa SL, eds. Functional Endocrine Pathology, 2nd ed. Boston: Blackwell Science, 1991:171–296. 48 Stanfield JP. The blood supply of the human pituitary gland. J Anat 1960;94:257–273. 49 Sheehan HL, Davis JC. Pituitary necrosis. Br Med Bull 1968;24:59–70. 50 Bergland RM, Page RB. Can the pituitary secrete directly to the brain? (Affirmative anatomical evidence). Endocrinology 1978;102:1325–1338. 51 Bergland RM, Page RB. Pituitary–brain vascular relations: a new paradigm. Science 1979;204:18–24.

52 Nieuwenhuys R. Chemoarchitecture of the brain. Berlin: Springer-Verlag, 1985. 53 Swaab DF, Fliers E. A sexually dimorphic nucleus in the human brain. Science 1985;228:1112–1115. 54 Gorski RA. Sexual differentiation of the brain. Hosp Pract 1978;13:55–62. 55 Whitehead R. The hypothalamus in post partum hypopituitarism. J Pathol Bacteriol 1963;86:55–67. 56 Cezayirli RC, Robertson JT. Pharmacologic modulation of hypothalamic control of appetite. In: Givens JR, ed. The Hypothalamus. Chicago: Year Book Medical Publishers Inc, 1984:115–128. 57 Liebelt RA, Perry JH. Action of gold thioglucose on the central nervous system. In: Handbook of Physiology, sect. 6, vol. 1. Alimentary canal control of food and water intake. Washington DC: American Physiological Society, 1967:271–285. 58 Sheehan HL. Le noyau subventriculaire de l’hypothalamus. Rev Fr Endocrinol Clin 1975;16:111–114. 59 Rance NE, McMullen NT, Smialek JE, Price DL, Young WS III. Postmenopausal hypertrophy of neurons expressing the estrogen receptor gene in the human hypothalamus. J Clin Endocrinol Metab 1990;71:79–85. 60 Ule G, Schwechheimer K, Tschahargane C. Morphological feedback effect on neurons of the nucl. arcuatus (sive infundibularis) and nucl. subventricularis hypothalami due to gonadal atrophy. Virchows Archiv (Pathol Anat) 1983;400:297–308. 61 Hart MN. Hypertrophy of human subventricular hypothalamic nucleus in starvation. Arch Pathol 1971;91:493–496. 62 Hommes OR. Effects of hypophysectomy and age on the infundibular nucleus in man. J Endocrinol 1974;62:479–488. 63 Sheehan HL, Kovacs K. The subventricular nucleus of the human hypothalamus. Brain 1966;89:589–614. 64 Sheehan HL. Variations in the subventricular nucleus. J Pathol Bacteriol 1967;94:409–416. 65 Weill J, Bernfeld J. Le Syndrome Hypothalamique. Paris: Masson & Cie, 1954. 66 Falin LI. The development of human hypophysis and differentiation of cells in its anterior lobe during embryonic life. Acta Anat 1961;44:188–205. 67 Hyyppä M. Hypothalamic monoamines in human fetuses. Neuroendocrinology 1972;9:257–266. 68 Goodyer CG, Guyda JH, Giroud CJP. Development of the hypothalamic– pituitary axis in the human fetus. In: Tolis G, Labrie F, Martin JB, Naftolin F, eds. Clinical Neuroendocrinology: a Pathophysiological Approach. New York: Raven Press, 1979:199–214. 69 Skowsky R, Fisher DA. Immunoreactive arginine vasopressin (AVP) and arginine vasotocin (AVT) in the fetal pituitary of man and sheep. J Clin Invest 1973;52:77a. 70 Benirschke K, McKay DG. The antidiuretic hormone in fetus and infant. Obstet Gynecol 1953;1:638–649. 71 Räiha N, Hjelt L. The correlation between the development of the hypophysial portal system and the onset of neurosecretory activity in the human fetus and infant. Acta Pediatr Scand 1957;72:610–616. 72 Rinne UK. Neurosecretory material passing into the hypophysial portal system in the human infundibulum, and its foetal development. Acta Neuroveg 1963;25:310–324. 73 Okado N, Yokota N. An electron microscopic study on the structural development of the neural lobe in the human fetus. Am J Anat 1980;159:261–273. 74 Bresson J-L, Clavequin M-C, Fellmann D et al. Ontogeny of the neuroglandular system revealed with HPGRF 44 antibodies in human hypothalamus. Neuroendocrinology 1984;39:68–73. 75 Bugnon C, Fellmann D, Bloch B. Immunocytochemical study of the ontogenesis of the hypothalamic somatostatin-containing neurons in the human fetus. Metabolism 1978;27(suppl. 1):1161–1165. 76 Ackland J, Ratter S, Bourne GL et al. Characterization of immunoreactive somatostatin in human fetal hypothalamic tissue. Regul Peptides 1983;5: 95–101. 77 Bugnon C, Fellmann D, Gouget A et al. Corticoliberin neurons: cytophysiology, phylogeny and ontogeny. J Steroid Biochem 1984;20:183–195. 78 Winters AJ, Eskay RL, Porter JC. Concentration and distribution of TRH and LRH in the human fetal brain. J Clin Endocrinol Metab 1974;39: 960–963. 79 Aubert ML, Grumbach MM, Kaplan SL. The ontogenesis of human fetal hormones. IV. Somatostatin, luteinizing hormone releasing factor, and thyrotropin releasing factor in hypothalamus and cerebral cortex of human fetuses 10–22 weeks of age. J Clin Endocrinol Metab 1977;44:1130–1141. 80 Bugnon C, Bloch B, Fellmann D. Cyto-immunological study of the ontogenesis of the gonadotropic hypothalamo–pituitary axis in the human fetus. J Steroid Biochem 1977;8:565–575.

Chapter 1 81 Siler-Khodr TM, Khodr GS. Studies in human fetal endocrinology. I. Luteinizing hormone-releasing factor content of the hypothalamus. Am J Obstet Gynecol 1978;130:795–800. 82 Bloch B, Brazeau P, Ling N et al. Immunohistochemical detection of growth hormone releasing factor in brain. Nature 1983;301:607–608. 83 Bloch B, Gaillard RC, Brazeau P et al. Topographical and ontogenetic study of the neurons producing growth hormone-releasing factor in human hypothalamus. Regul Peptides 1984;8:21–31. 84 Bloch B, Brazeau P, Bloom F et al. Topographical study of the neurons containing hpGRF immunoreactivity in monkey hypothalamus. Neurosci Lett 1983;37:23–28. 85 Merchenthaler I, Vigh S, Schally AV et al. Immunocytochemical localization of growth hormone-releasing factor in the rat hypothalamus. Endocrinology 1984;114:1082–1085. 86 Vaccarino FJ, Bloom FE, Rivier J et al. Stimulation of food intake in rats by centrally administered hypothalamic growth hormone-releasing factor. Nature 1985;314:167–168. 87 Sano T, Asa SL, Kovacs K. Growth hormone-releasing hormone-producing tumors: clinical, biochemical and morphological manifestations. Endocr Rev 1988;9:357–373. 88 Krulich L, Dhariwal APS, McCann S. Stimulatory and inhibitory effects of purified hypothalamic extracts on growth hormone release from rat pituitary in vitro. Endocrinology 1968;83:783–790. 89 Elde RP, Parsons JA. Immunocytochemical localization of somatostatin in cell bodies of the rat hypothalamus. Am J Anat 1975;144:541–548. 90 Désy L, Pelletier G. Immunohistochemical localization of somatostatin in the human hypothalamus. Cell Tissue Res 1977;184:491–497. 91 Finley JCW, Maderchut JL, Roger LJ et al. The immunocytochemical localization of somatostatin-containing neurons in the rat central nervous system. Neuroscience 1981;6:2173–2192. 92 Pelletier G, Dubé D, Puviani R. Somatostatin. Electron microscopic immunohistochemical localization in secretory neurons of rat hypothalamus. Science 1977;196:1496–1470. 93 Hökfelt T, Efendic S, Hellerstrom C et al. Cellular localization of somatostatin in endocrine-like cells and neurons of the rat with special references to the A1-cells of the pancreatic islets and to the hypothalamus. Acta Endocrinol 1975;80(suppl.):5–41. 94 Reichlin S. Somatostatin. Part I. N Engl J Med 1983;309:1495–1501. 95 Reichlin S. Somatostatin. Part II. N Engl J Med 1983;309:1556–1563. 96 Ben Jonathan N. Dopamine: a prolactin-inhibiting hormone. Endocr Rev 1985;6:564–589. 97 Bjorklund A, Lindvall O, Nobin A. Evidence of an incertohypothalamic dopamine neuron system in the rat. Brain Res 1975;89:29–42. 98 Ajika K, Hökfelt T. Projections to the median eminence and the arcuate nucleus, with special reference to monoamine systems: effects of lesions. Cell Tissue Res 1975;158:15–35. 99 Olschowka JA, O’Donohue TL, Mueller GP et al. The distribution of corticotropin releasing factor-like immunoreactive neurons in rat brain. Peptides 1982;3:995–1015. 100 Merchenthaler I, Vigh S, Petrusz P et al. Immunocytochemical localization of corticotropin-releasing factor (CRF) in the rat brain. Am J Anat 1982;165: 385–396. 101 Swanson JW, Sawchenko PE, Rivier J et al. Organization of ovine corticotropin-releasing factor immunoreactive cells and fibers in the rat brain: an immunohistochemical study. Neuroendocrinology 1983;36: 165–186. 102 Pelletier G, Désy L, Côté J et al. Immunocytochemical localization of corticotropin-releasing factor-like immunoreactivity in the human hypothalamus. Neurosci Lett 1983;41:259–263. 103 Roth KA, Weber E, Barchas JD. Immunoreactive corticotropin-releasing factor (CRF) and vasopressin are colocalized in a subpopulation of the immunoreactive vasopressin cells in the paraventricular nucleus of the hypothalamus. Life Sci 1982;31:1857–1860. 104 Sawchenko PE, Swanson LW. Localization, colocalization, and plasticity of corticotropin-releasing factor immunoreactivity in rat brain. Fed Proc 1985;44:221–227. 105 Petrusz P, Merchenthaler I, Maderdrut JL et al. Central and peripheral distribution of corticotropin-releasing factor. Fed Proc 1985;44: 229–235. 106 Audhya T, Hollander CS, Schlesinger DH et al. Structural characterization and localization of corticotropin-releasing factor in testis. Biochim Biophys Acta 1989;995:10–16. 107 Brownstein MJ, Palkovits M, Saavedra JM et al. Thyrotropin-releasing hormone in specific nuclei of the brain. Science 1974;185:267–269.

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108 Hökfelt T, Fuxe K, Johansson O et al. Thyrotropin-releasing hormone (TRH) in the central nervous system as revealed with immunohistochemistry. Eur J Pharmacol 1975;34:389–392. 109 Horita A, Carino MA, Chestnut RM. Influence of TRH on drug-induced narcosis and hyperthermia in the rabbit. Psychopharmacology 1976;49:57–62. 110 Kawano H, Daikoku S, Saito S. Location of thyrotropin-releasing hormonelike immunoreactivity in rat pancreas. Endocrinology 1983;112:951–955. 111 Dolva LO, Hanssen KF, Aadland E et al. Thyrotropin-releasing hormone immunoreactivity in the gastrointestinal tract of man. J Clin Endocrinol Metab 1983;56:524–529. 112 Wilber JF, Spinella P. Identification of immunoreactive thryotropin-releasing hormone in human neoplasia. J Clin Endocrinol Metab 1984;59:432–435. 113 Baker BL, Dermody WC, Reell JR. Localization of luteinizing hormonreleasing hormone in the mammalian hypothalamus. Am J Anat 1974;139: 129–134. 114 Barry J, Dubois MP, Poulain P. LRF-producing cell of the mammalian hypothalamus. Z Zellforsch 1974;146:351–366. 115 Pelletier G, Labrie F, Puviani R et al. Immunohistochemical localization of luteinizing hormone-releasing hormone in the rat median eminence. Endocrinology 1974;1:157–166. 116 Siler-Khodr TM, Khodr GS. Luteinizing hormone releasing factor content of the human placenta. Am J Obstet Gynecol 1978;130:216–219. 117 Dierickx K, Vandesande F. Immunocytochemical demonstration of separate vasopressin–neurophysin and oxytocin–neurophysin neurons in the human hypothalamus. Cell Tissue Res 1979;196:203–212. 118 Antunes JL, Carmel PW, Zimmerman EA. Paraventricular nucleus: the origin of vasopressin secreting terminals in hypophysial portal vessels in the monkey. Brain Res 1977;137:1–10. 119 Lederis K. An electron microscopical study of the human neurohypophysis. Z Zellforsch 1965;65:847–868. 120 Silverman AJ. Ultrastructural studies on the localization of neurohypophysial hormones and their carrier proteins. J Histochem Cytochem 1976;24: 816–827. 121 Stillman MA, Recht LD, Rosario SL et al. The effects of adrenalectomy glucocorticoid replacement of vasopressin and vasopressin–neurophysin in the zona externa of the median eminence of the rat. Endocrinology 1977;101: 42–49. 122 Dubé D, Leclerc R, Pelletier G. Electron microscopic immunohistochemical localization of vasopressin and neurophysin in the median eminence of normal and adrenalectomized rat. Am J Anat 1976;147:103–108. 123 Clements JA, Funder JW. Arginine vasopressin (AVP) and AVP-like immunoreactivity in peripheral tissues. Endocr Rev 1986;7:449–460. 124 Polak JM, Bloom SR. Distribution and tissue localization of VIP in the central nervous system and seven peripheral organs. In: Said S, ed. Vasoactive Intestinal Peptide. New York: Raven Press, 1982:107–120. 125 Hökfelt T, Fahrenkrug J, Ju G et al. Analysis of peptide histidineisoleucine/vasoactive intestinal polypeptide-immunoreactive neurons in the central nervous system with special reference to their relation to corticotropin releasing factor- and enkephalin-like immunoreactivities in the paraventricular hypothalamic nucleus. Neuroscience 1987;23:827–857. 126 Watanabe H. The immunostaining for the hypothalamic vasoactive intestinal peptide but not for beta-endorphin, dynorphin-A or methionine-enkephalin, is affected by the glucocorticoid milieu in the rat: correlation with the prolactin secretion. Regulatory Peptides 1990;28:301–311. 127 Prysor-Jones RA, Jenkins JS. Vasoactive intestinal peptide and anterior pituitary function. Clin Endocrinol 1988;29:677–688. 128 Vijaryan E, Samson WK, Saide SD et al. Vasoactive intestinal peptide: evidence for a hypothalamic site of action to release growth hormone, luteinizing hormone and prolactin in conscious ovariectomized rats. Endocrinology 1979;104:53–57. 129 Vanderhaeghen JJ, Signeau JC, Gepts W. New peptide in the vertebrate CNS reacting with gastrin antibodies. Nature 1978;257:604–605. 130 Crowley JN. Comparative distribution of cholecystokinin and other neuropeptides. Ann NY Acad Sci 1985;448:1–8. 131 Hökfelt T, Herrera-Marschitz M, Seroogy K et al. Immunohistochemical studies on cholecystokinin (CCK)-immunoreactive neurons in the rat using sequence specific antisera with special reference to the caudate nucleus and primary sensory neurons. J Chem Neuroanat 1988;1:11–51. 132 Albus M. Cholecystokinin. Prog Neuro-Psychopharmacol Biol Psych 1988; 12(suppl.):S5–21. 133 Tatemoto K, Rokaeus A, Jornvall H et al. Galanin—a novel biologically active peptide from porcine intestine. FEBS Lett 1983;164:124–128. 134 Skofitsch G, Jacobowitz DM. Immunohistochemical mapping of galanin-like neurons in the rat central nervous system. Peptides 1985;6:509–546.

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Hypothalamic–Pituitary Function

135 Melander T, Hökfelt T, Rokaeus A et al. Coexistence of galanin-like immunoreactivity with catecholamines, 5-OH-tryptamine, GABA and neuropeptides in the rat CNS. Neuroscience 1986;6:3640–3656. 136 Cheung A, Polak JM, Bauer FE et al. The distribution of galanin immunoreactivity in the respiratory tract in pig, guinea-pig, rat and dog. Thorax 1985;40:889–896. 137 Bauer FE, Christofides ND, Hacker GW et al. Distribution of galanin immunoreactivity in the genitourinary tract of man and rat. Peptides 1986;7:5–10. 138 Bauer FE, Hacker GW, Terenghi G et al. Localization and molecular forms of galanin in human adrenals: elevated levels in pheochromocytomas. J Clin Endocrinol Metab 1986;63:1372–1378. 139 Sano T, Vrontakis ME, Kovacs K et al. Galanin immunoreactivity in neuroendocrine tumors. Arch Pathol Lab Med 1991;115:926–929. 140 Nordström O, Melander T, Hökfelt T et al. Evidence for an inhibitory effect of the peptide galanin on dopamine release from the rat median eminence. Neurosci Lett 1987;73:21–26. 141 Murakami Y, Kato Y, Shimatsu A et al. Possible mechanisms involved in growth hormone secretion induced by galanin in the rat. Endocrinology 1989;124:1224–1229. 142 Rehfeld JF. Localization of gastrins to neuro- and adenohypophysis. Nature 1978;271:771–773. 143 Rehfeld JF, Hansen HF, Marley PD et al. Molecular forms of cholecystokinin in the brain and the relationship to neuronal gastrins. Ann NY Acad Sci 1985;448:11–23. 144 McCann SM. The role of brain peptides in the control of anterior pituitary hormone secretion. In: Müller EE, MacLeod RM, eds. Neuroendocrine Perspectives, vol. 1. New York: Elsevier, 1982:1–22. 145 Lorén I, Alumets J, Ha° kanson R, Sundler F, Thorell J. Gut-type glucagon immunoreactivity in nerves of the rat brain. Histochemistry 1979;61: 335–341. 146 Dorn A, Rinne A, Bernstein HG et al. Immunoreactive glucagon in neurons of various parts of the human brain. Demonstration by immunofluorescence technique. Acta Histochem 1981;69:243–247. 147 Drucker DJ, Asa SL. Glucagon gene expression in vertebrate brain. J Biol Chem 1988;263:13475–13478. 148 Inokuchi A, Oomura Y, Shimizu N et al. Central action of glucagon in rat hypothalamus. Am J Physiol 1986;250:R120–126. 149 Allen YS, Adrian TE, Allen JM et al. Neuropeptide Y distribution in the rat brain. Science 1983;221:8877–8879. 150 Danger JM, Tonon MC, Jenks BG et al. Neuropeptide Y: localization in the central nervous system and neuroendocrine functions. Fundam Clin Pharmacol 1990;4:307–340. 151 Toni R, Jackson IMD, Lechan RM. Neuropeptide Y-immunoreactive innervation of thyrotropin-releasing-hormone-synthesizing neurons in the rat hypothalamic paraventricular nucleus. Endocrinology 1990;126:2444–2453. 152 Aronin N, Coslovsky R, Leeman SE. Substance P and neurotensin: their roles in the regulation of anterior pituitary function. Annu Rev Physiol 1986;48:537–549. 153 Carraway R, Leeman S. Characterization of radioimmunoassayable neurotensin in the rat: its differential distribution in the central nervous system, small intestine and stomach. J Biol Chem 1976;251:7045–7052. 154 Panula P, Hsiu-Ying TY, Costa E. Comparative distribution of bombesin/GRP- and substance P-like immunoreactivities in rat hypothalamus. J Comp Neurol 1984;224:606–618. 155 Giraud AS, Smith AI, Rundle SE. Distribution and molecular forms of immunoreactive bombesin in the ovine median eminence. Mol Cell Endocrinol 1987;53:245–250. 156 Takahashi K, Mouri T, Sone M et al. Calcitonin gene-related peptide in the human hypothalamus. Endocrinol Jpn 1989;36:409–415. 157 Fahim A, Rettori V, McCann SM. The role of calcitonin gene-related peptide on the control of growth hormone and prolactin release. Neuroendocrinology 1990;51:688–693. 158 Smith AI, Funder JW. Proopiomelanocortin processing in the pituitary, central nervous system, and peripheral tissues. Endocr Rev 1988;9:159–179. 159 Plosker SM, Marshall LA, Martin MC, Jaffe RB. Opioid, catecholamine, and steroid interaction in prolactin and gonadotropin regulation. Obstet Gynecol Surv 1990;45:441–453. 160 Zhao BG, Chapman C, Brown D, Bicknell RJ. Opioid––noradrenergic interactions in the neurohypophysis. II. Does noradrenaline mediate the actions of endogenous opioids on oxytocin and vasopressin release? Neuroendocrinology 1988;48:25–31. 161 Bateman A, Singh A, Kral T, Solomon S. The immune–hypothalamic– pituitary–adrenal axis. Endocr Rev 1989;10:92–112.

162 Bernton EW, Beach JE, Holaday JW et al. Release of multiple hormones by a direct action of interleukin-1 on pituitary cells. Science 1987;238:519–524. 163 Scarborough DE, Lee SL, Dinarello CA, Reichlin S. Interleukin-1b stimulates somatostatin biosynthesis in primary cultures of fetal rat brain. Endocrinology 1989;124:549–551. 164 Spangelo BL, Judd AM, Isakson PC, MacLeod RM. Interleukin-6 stimulates anterior pituitary hormone release in vitro. Endocrinology 1989;125:575–577. 165 Yamaguchi M, Sakata M, Matsuzaki N et al. Induction by tumor necrosis factor-alpha of rapid release of immunoreactive and bioactive luteinizing hormone from rat pituitary cells in vitro. Neuroendocrinology 1990;52: 468–472. 166 Walton PE, Cronin MJ. Tumor necrosis factor-a and interferon-gamma reduce prolactin release in vitro. Am J Physiol 1990;259:E672–676. 167 Rizzino A. Growth factors. In: Kovacs K, Asa SL, eds. Functional Endocrine Pathology. Boston: Blackwell Science, 1991:979–989. 168 Gospodarowicz D, Cheng J, Lui G-M et al. Isolation of brain fibroblast growth factor by heparin–sepharose affinity chromatography: identity with pituitary fibroblast growth factor. Proc Natl Acad Sci USA 1984;81: 6963–6967. 169 Kudlow JE, Leung AWC, Kobrin MS et al. Transforming growth factor-a in the mammalian brain. Immunohistochemical detection in neurons and characterization of its mRNA. J Biol Chem 1989;264:3880–3883. 170 Baird A, Mormede P, Ying SY et al. A nonmitogenic pituitary function of fibroblast growth factor: regulation of thyrotropin and prolactin secretion. Proc Natl Acad Sci USA 1985;82:5545–5549. 171 Ikeda H, Mitsuhashi T, Kubota K et al. Epidermal growth factor stimulates growth hormone secretion from superfused rat adenohypophyseal fragments. Endocrinology 1984;115:556–558. 172 Schriock EA, Lustig RH, Rosenthal SM et al. Effect of growth hormone (GH)-releasing hormone (GHRH) on plasma GH in relation to magnitude and duration of GH deficiency in 26 children and adults with isolated GH deficiency or multiple pituitary hormone deficiencies: evidence for hypothalamic GRH deficiency. J Clin Endocrinol Metab 1984;58:1043–1049. 173 Rimoin DL. Hereditary forms of growth hormone deficiency and resistance. Birth Defects 1976;12:15–29. 174 Phillips JA III, Parks JS, Hjelle BL et al. Genetic analysis of familial isolated growth hormone deficiency type I. J Clin Invest 1982;70:489–495. 175 Rimoin DL, Schechter JE. Histologic and ultrastructural studies in isolated growth hormone deficiency. J Clin Endocrinol Metab 1973;37:725–735. 176 Santora N, Filicori M, Crowley WF Jr. Hypogonadotropic disorders in men and women: diagnosis and therapy with pulsatile gonadotropin-releasing hormone. Endocr Rev 1986;7:11–23. 177 Toledo SP, Luthold W, Mattar E. Familial idiopathic gonadotropin deficiency: a hypothalamic form of hypogonadism. Am J Med Genet 1983;15:405–416. 178 Stocks AE, Martin FIR. Pituitary function in hemochromatosis. Am J Med 1968;45:839–845. 179 Soules MR, Hammond CB. Female Kallmann’s syndrome: evidence for a hypothalamic luteinizing hormone-releasing hormone deficiency. Fertil Steril 1980;33:82–85. 180 Klingmüller D, Dewes W, Krahe T et al. Magnetic resonance imaging of the brain in patients with anosmia and hypothalamic hypogonadism (Kallmann’s syndrome). J Clin Endocrinol Metab 1987;65:581–584. 181 Seminara SB, Hayes FJ, Crowley WF Jr. Gonadotropin-releasing hormone deficiency in the human (idiopathic hypogonadotropic hypogonadism and Kallman’s syndrome): pathophysiological and genetic considerations. Endocrine Rev 1998;19:521–539. 182 Kovacs K, Sheehan HL. Pituitary changes in Kallmann’s syndrome: a histologic, immunocytologic, ultrastructural, and immunoelectron microscopic study. Fertil Steril 1982;37:83–89. 183 Daniel PM, Prichard MML. Studies of the hypothalamus and the pituitary gland: with special reference to the effects of transection of the pituitary stalk. Acta Endocrinol 1975;201(suppl.):1–216. 184 Heinbecker P, White HL. Hypothalamico-hypophysial system and its relation to water balance in the dog. Am J Physiol 1941;133:582–593. 185 Green JR, Buchan GC, Alvord EC Jr, Swanson AG. Hereditary and idiopathic types of diabetes insipidus. Brain 1967;90:707–714. 186 Nagai I, Li CH, Hsieh SM et al. Two cases of hereditary diabetes insipidus, with an autopsy finding in one. Acta Endocrinol 1984;105:318–323. 187 Scherbaum WA, Bottazzo GF. Autoantibodies to vasopressin cells in idiopathic diabetes insipidus: evidence for an autoimmune variant. Lancet 1983;i: 897–901. 188 Melmed S, Ezrin C, Kovacs K et al. Acromegaly due to secretion of growth hormone by an ectopic pancreatic islet-cell tumor. N Engl J Med 1985;312: 9–17.

Chapter 1 189 Asa SL, Scheithauer BW, Bilbao JM et al. A case for hypothalamic acromegaly: a clinicopathologic study of six patients with hypothalamic gangliocytomas producing growth hormone-releasing factor. J Clin Endocrinol Metab 1984;58:796–803. 190 Kovacs K, Horvath E, Thorner MO, Rogol AD. Mammosomatotroph hyperplasia associated with acromegaly and hyperprolactinemia in a patient with the McCune–Albright syndrome: a histologic, immunocytologic and ultrastructural study of the surgically-removed adenophypophysis. Virchows Archiv (Pathol Anat) 1984;403:77–86. 191 Weinstein LS, Shenker A, Gejman PV et al. Activating mutations of the stimulatory G protein in the McCune–Albright syndrome. New Engl J Med 1991;325:1688–1695. 192 Schwindinger WF, Francomano CA, Levine MA. Identification of a mutation in the gene encoding the alpha subunit of the stimulatory G protein of adenylyl cyclase in McCune–Albright syndrome. Proc Natl Acad Sci USA 1992;89:5152–5156. 193 Moran A, Asa SL, Kovacs K et al. Pituitary mammosomatotroph hyperplasia associated with childhood gigantism. New Engl J Med 1990;323:322–327. 194 Stefaneanu S, Kovacs K, Horvath E et al. Adenohypophysial changes in mice transgenic for human growth hormone-releasing factor: a histologic, immunocytochemical and electron microscopic investigation. Endocrinology 1989;125:2710–2718. 195 Asa SL. Tumors of the Pituitary Gland. Atlas of Tumor Pathology, Fascicle 22, Third Series. Washington DC: Armed Forces Institute of Pathology, 1998. 196 Krieger DT. Pathophysiology of Cushing’s Disease. Endocr Rev 1983;4:22–43. 197 Carey RM, Varma SK, Drake CR Jr et al. Ectopic secretion of corticotropinreleasing factor as a cause of Cushing’s syndrome: a clinical, morphologic, and biochemical study. N Engl J Med 1984;311:13–20. 198 Asa SL, Kovacs K, Vale W et al. Immunohistologic localization of corticotropin-releasing hormone in human tumors. Am J Clin Pathol 1987;87: 327–333. 199 Asa SL, Kovacs K, Tindall GT et al. Cushing’s disease associated with an intrasellar gangliocytoma producing corticotropin-releasing factor. Ann Intern Med 1984;101:789–793. 200 Puchner MJA, Lüdecke DK, Valdueza JM et al. Cushing’s disease in a child caused by a corticotropin-releasing hormone-secreting intrasellar gangliocytoma associated with an adrenocorticotropic hormone-secreting pituitary adenoma. Neurosurgery 1993;33:920–925. 201 Puchner MJA, Lüdecke DK, Saeger W et al. Gangliocytomas of the sellar region—a review. Exp Clin Endocrinol 1995;103:129–149. 202 Liu N, Grumbach MM, de Napoli RA, Morishima A. Prevalence of electroencephalographic abnormalities in idiopathic precocious puberty and premature pubarche: bearing on pathogenesis and neuroendocrine regulation of puberty. J Clin Endocrinol Metab 1965;25:1296–1308. 203 Rosenthal SM, Grumbach MM, Kaplan SL. Gonadotropin-independent familial sexual precocity with premature Leydig and germinal cell maturation (familial testotoxicosis): effects of a potent luteinizing hormone-releasing factor agonist and medroxyprogesterone acetate therapy in four cases. J Clin Endocrinol Metab 1983;57:571–579. 204 Judge DM, Kulin HE, Page R et al. Hypothalamic hamartoma: a source of luteinizing-hormone-releasing factor in precocious puberty. N Engl J Med 1977;296:7–10. 205 Hochman HI, Judge DM, Reichlin S. Precocious puberty and hypothalamic hamartoma. Pediatrics 1981;67:236–244. 206 Kamoi K, Mitsuma T, Sato H et al. Hyperthyroidism caused by a pituitary thyrotrophin-secreting tumor with excessive secretion of thyrotrophinreleasing hormone and subsequently followed by Grave’s disease in a middle aged woman. Acta Endocrinol 1985;110:373–382. 207 DeTroyer A, Demanet JC. Clinical, biological and pathogenic features of the syndrome of inappropriate secretion of antidiuretic hormone: a review of 26 cases with marked hyponatraemia. Q J Med 1976;45:521–531. 208 Vorherr H, Massry SG, Fallet R et al. Antidiuretic principle in tuberculous lung tissue of a patient with pulmonary tuberculosis and hyponatremia. Ann Intern Med 1970;72:383–387. 209 Pettengill OS, Faulkner CS, Wurster-Hill DH et al. Isolation and characterization of a hormone-producing cell line from human small cell anaplastic carcinoma of the lung. J Natl Cancer Inst 1977;58:511–518. 210 Bergeron C, Kovacs K, Bilbao JM. Primary empty sella. A histologic and immunocytologic study. Arch Intern Med 1979;139:248–249. 211 Bergland RM, Ray BS, Torack RM. Anatomical variations in the pituitary gland and adjacent structures in 225 human autopsy cases. J Neurosurg 1968;28:93–99. 212 Gorczyca W, Hardy J. Arterial supply of the human anterior pituitary gland. Neurosurgery 1987;20:369–378.

Functional Anatomy of the Hypothalamic Pituitary Axis

41

213 Cox BM, Baizman ER, Su T-P et al. Further studies on the nature and function of pituitary endorphins. Adv Biochem Psychopharmacol 1978;18: 183–189. 214 Ferrand R, Hraoui S. Origine exlusivement ectodermique de l’adénohypophyse chez la caille et le poulet; demonstration par la méthode des associations tissulaires interspécifiques. C R Soc Biol 1973;167:740–743. 215 Ferrand R, Pearse AGE, Polak JM, Le Douarin NM. Immunohistochemical studies on the development of avian embryo pituitary corticotrophs under normal and experimental conditions. Histochemistry 1974;38:133–141. 216 LeDouarin N. Particularités du noyau interphasique chez la caille japonaise (Coturnix coturnix japonica). Utilisation de ces particularités comme “marque biologique” dans les recherches sur les interactions tissulaires et les migrations cellulaires au cours de l’ontogénèse. Bull Biol Fr Belg 1969;103: 435–452. 217 Takor Takor T, Pearse AGE. Neuroectodermal origin of avian hypothalamohypophyseal complex: the role of the ventral neural ridge. J Embryol Exp Morphol 1975;34:311–325. 218 Daikoku S. Studies on the human foetal pituitary. 1. Quantitative observations. Tokushima J Exp Med 1958;5:200–213. 219 Baker BL, Jaffe RB. The genesis of cell types in the adenohypophysis of the human fetus as observed by immunocytochemistry. Am J Anat 1975;143: 137–162. 220 Asa SL, Kovacs K, Laszlo FA et al. Human fetal adenohypophysis. Histologic and immunocytochemical analysis. Neuroendocrinology 1986;43:308–316. 221 Asa SL, Kovacs K, Horvath E et al. Human fetal adenohypophysis. Electron microscopic and ultrastructural immunocytochemical analysis. Neuroendocrinology 1988;48:423–431. 222 Tremblay JJ, Lanctot C, Drouin J. The pan-pituitary activator of transcription, Ptxl (pituitary homeobox 1) acts in synergy with SF-1 and Pit-1 and is an upstream regulator of the Lim-homeodomain gene Lim3/Lhx3. Mol Endocrinol 1998;12:428–441. 223 Gage PJ, Camper SA. Pituitary Homeobox 2, a novel member of the bicoidrelated family of homeobox genes, is a potential regulator of anterior structure formation. Hum Mol Genet 1997;6:457–464. 224 Sheng HZ, Moriyama K, Yamashita T et al. Multistep control of pituitary organogenesis. Science 1997;278:1809–1812. 225 Bach I, Rhodes SJ, Pearse RV et al. P-Lim, a LIM homeodomain factor, is expressed during pituitary organ and cell commitment and synergizes with Pit-1. Proc Natl Acad Sci USA 1995;92:2720–2724. 226 Hermesz E, Machem S, Mahon KA. Rpx: a novel anterior-restricted homeobox gene progressively activated in the prechordal plate, anterior neural plate and Rathke’s pouch of the mouse embryo. Develop 1996;122:41–52. 227 Sornson MW, Wu W, Dasen JS et al. Pituitary lineage determination by the Prophet of Pit-1 homeodomain factor defective in Ames dwarfism. Nature 1996;384:327–333. 228 Gage PJ, Brinkmeier MLSLM, Knapp LT. The Ames dwarf gene, df, is required early in pituitary ontogeny for the extinction of Rpx transcription and initiation of lineage-specific cell proliferation. Mol Endocrinol 1996;10: 1570–1581. 229 Wu W, Cogan JD, Pfäffle RW et al. Mutations in PROP1 cause familial combined pituitary hormone deficiency. Nature Genet 1998;18:147–149. 230 Fofanova O, Takmura N, Kinoshita E. Compound heterozygous deletion of the prop-1 gene in children with combined pituitary hormone deficiency. J Clin Endocrinol Metab 1998;83:2601–2604. 231 Jackson SM, Barnhart KM, Mellon P et al. Helix-loop proteins are present and differentially expressed in different cell lines from the anterior pituitary. Mol Cell Endocrinol 1993;96:167–176. 232 Asa SL, Ezzat S. The cytogenesis and pathogenesis of pituitary adenomas. Endocr Rev 1998;19:798–827. 233 Asa SL, Ezzat S. Molecular determinants of pituitary cytodifferentiation. Pituitary 1999;1:159–168. 234 Lamolet B, Pulichino AM, Lamonerie T et al. A pituitary cell-restricted T box factor, T pit, activates POMC transcription in cooperation with Pitx homeoproteins. Cell 2001;104:849–859. 235 Poulin G, Turgeon B, Drouin J. NeuroD1/beta2 contributes to cell-specific transcription of the proopiomelanocortin gene. Mol Cell Biol 1997;17: 6673–6682. 236 Ingraham HA, Lala DS, Ikeda Y et al. The nuclear receptor steroidogenic factor 1 acts at multiple levels of the reproductive axis. Genes Dev 1994;8:2302–2312. 237 Asa SL, Bamberger A-M, Cao B et al. The transcription activator steroidogenic factor-1 is preferentially expressed in the human pituitary gonadotroph. J Clin Endocrinol Metab 1996;81:2165–2170.

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SECTION 1

Hypothalamic–Pituitary Function

238 Rosenfeld MG. POU-domain transcription factors: pou-er-ful developmental regulators. Genes Dev 1991;5:897–907. 239 Asa SL, Puy LA, Lew AM et al. Cell type-specific expression of the pituitary transcription activator Pit-1 in the human pituitary and pituitary adenomas. J Clin Endocrinol Metab 1993;77:1275–1280. 240 Friend KE, Chiou Y-K, Laws ER Jr et al. Pit-1 messenger ribonucleic acid is differentially expressed in human pituitary adenomas. J Clin Endocrinol Metab 1993;77:1281–1286. 241 Pellegrini I, Barlier A, Gunz G et al. Pit-1 gene expression in the human pituitary and pituitary adenomas. J Clin Endocrinol Metab 1994;79:189–196. 242 Day RN, Koike S, Sakai M et al. Both Pit-1 and the estrogen receptor are required for estrogen responsiveness of the rat prolactin gene. Mol Endocrinol 1990;4:1964–1971. 243 Drolet DW, Scully KM, Simmons DM et al. TEF, a transcription factor expressed specifically in the anterior pituitary during embryogenesis, defines a new class of leucine zipper proteins. Genes Dev 1991;5:1739–1753. 244 Li S, Crenshaw EB III, Rawson EJ et al. Dwarf locus mutants lacking three pituitary cell types result from mutations in the POU-domain gene pit-1. Nature 1990;347:528–533. 245 Tatsumi K, Miyai K, Notomi T et al. Cretinism with combined hormone deficiency caused by a mutation in the Pit-1 gene. Nature Genet 1992;1:56–58. 246 Pfäffle RW, DiMattia GE, Parks JS et al. Mutation of the POU-specific domain of Pit-1 and hypopituitarism without pituitary hypoplasia. Science 1992;257:1118–1121. 247 Radovick S, Nations M, Du Y et al. A mutation in the POU-homeodomain of Pit-1 responsible for combined pituitary hormone deficiency. Science 1992;257:1115–1118. 248 Puy LA, Asa SL. The ontogeny of pit-1 expression in the human fetal pituitary gland. Neuroendocrinology 1996;63:349–355. 249 Ikeda Y, Luo X, Abbud R et al. The nuclear receptor steroidogenic factor 1 is essential for the formation of the ventromedial hypothalamic nucleus. Mol Endocrinol 1995;9:478–486. 250 Yang H-J, Ozawa H, Kurosumi K. Ultrastructural changes in growth hormone cells in the rat anterior pituitary after thyroidectomy as studied by immunoelectron microscopy and enzyme nistochemistry. J Clin Electr Microsc 1989;22:269–283. 251 Ezzat S, Kontogeorgos G, Redelmeier DA et al. In vitro responsiveness of morphological variants of growth hormone-producing pituitary adenomas to octreotide. Eur J Endocrinol 1995;133:686–690. 252 Horvath E, Kovacs K. The adenohypophysis. In: Kovacs K, Asa SL, eds. Functional Endocrine Pathology 2nd ed. Boston: Blackwell Science, 1998; 247–281. 253 Sano T, Ohshima T, Yanad S. Expression of glycoprotein hormones and intracytoplasmic distribution of cytokeratin on growth hormone-producing pituitary adenomas. Pathol Res Pract 1991;187:530–533. 254 Neumann PE, Goldman JE, Horoupian DS, Hess MA. Fibrous bodies in growth hormone-secreting adenomas contain cytokeratin filaments. Arch Pathol Lab Med 1985;109:505–508. 255 Billestrup N, Swanson L, Vale W. Growth hormone-releasing factor stimulates proliferation of somatotrophs in vitro. Proc Natl Acad Sci USA 1986;83: 6854–6857. 256 Richardson SB, Twente S. Evidence that diminished pituitary responsitivity to GHRF is secondary to intracellular GH pool depletion. Am J Physiol 1988;254:E358–364. 257 Loras B, Yi JY, Durand A et al. GRF et adénomes somatotropes humains. Correlations in vivo et in vitro entre la libération de GH et les aspects morphologiques et immunocytochimiques. Ann Endocrinol 1985;46: 373–382. 258 Kawakita S, Asa SL, Kovacs K. Effects of growth hormone-releasing hormone (GHRH) on densely granulated somatotroph adenomas and sparsely granulated somatotroph adenomas in vitro: a morphological and functional investigation. J Endocrinol Invest 1989;12:443–448. 259 Beckers A, Stevenaert A, Kovacs K et al. The treatment of acromegaly with SMS 201–995. Advanc Biosci 1988;69:227–228. 260 Barkan AL, Lloyd RV, Chandler WF et al. Preoperative treatment of acromegaly with long-acting somatostatin analog SMS 201–995: shrinkage of invasive pituitary macroadenomas and improved surgical remission rate. J Clin Endocrinol Metab 1988;67:1040–1048. 261 Ezzat S, Snyder PJ, Young WF et al. Octreotide treatment of acromegaly. A randomized, multicenter study. Ann Intern Med 1992;117:711–718. 262 Asa SL, Felix I, Kovacs K, Ramyar L. Effects of somatostatin on somatotroph adenomas of the human pituitary: an in vitro functional and morphological study. Endocr Pathol 1990;1:228–235.

263 Baringa M, Bilezikjian LM, Vale WW, Rosenfeld MG, Evans RM. Independent effects of growth hormone releasing factor on growth hormone release and gene transcription. Nature 1985;314:279–281. 264 Herman V, Weiss M, Becker D, Melmed S. Hypothalamic hormonal regulation of human growth hormone gene expression in somatotroph adenoma cell cultures. Endocr Pathol 1990;1:236–244. 265a Zafar M, Ezzat S, Ramyar L et al. Cell specific expression of estrogen receptor in the human pituitary and its adenomas. J Clin Endocrinol Metab 1995;80:3621–3627. 265b Friend KE, Chiou TK, Lopes MBS et al. Estrogen receptor expression in human pituitary. J Clin Endocrinol Metab 1994;78:1497–1504. 266 Asa SL, Penz G, Kovacs K, Ezrin C. Prolactin cells in the human pituitary: a quantitative immunocytochemical analysis. Arch Pathol Lab Med 1982;106: 360–363. 267 Scheithauer BW, Sano T, Kovacs K et al. The pituitary gland in pregnancy: a clinicopathologic and immunohistochemical study of 69 cases. Mayo Clin Proc 1990;65:461–474. 268a Scheithauer BW, Kovacs K, Randall RV, Ryan N. Effects of estrogen on the human pituitary: a clinicopathologic study. Mayo Clin Proc 1989;64: 1077–1084. 268b Asa SL, Kelly MA, Grandy DK, Low MJ. Pituitary lactotroph adenomas develop after prolonged lactotroph hyperplasia in dopamine D2 receptordeficient mice. Endocrinology 1999;140:5348–5355. 269 Tindall GT, Kovacs K, Horvath E, Thorner MO. Human prolactin-producing adenomas and bromocriptine: a histological, immunocytochemical, ultrastructural, and morphometric study. J Clin Endocrinol Metab 1982;55: 1178–1183. 270 Esiri MM, Bevan JS, Burke CW, Adams CBI. Effect of bromocriptine treatment on the fibrous tissue component of prolactin-secreting and nonfunctioning macroadenomas of the pituitary gland. J Clin Endocrinol Metab 1986;63:383–388. 271 Felix IA, Horvath E, Kovacs K et al. Mammosomatotroph adenoma of the pituitary associated with gigantism and hyperprolactinemia. Acta Neuropathol 1986;71:76–82. 272 Asa SL, Kovacs K, Stefaneanu L et al. Pituitary adenomas in mice transgenic for growth hormone-releasing hormone. Endocrinology 1992;131:2083–2089. 273 Lewis ME, Sherman TG, Burke S et al. Detection of proopiomelanocortin mRNA by in situ hybridization with an oligonucleotide probe. Proc Natl Acad Sci USA 1986;83:5419–5423. 274 Horvath E, Ilse G, Kovacs K. Enigmatic bodies in human corticotroph cells. Acta Anat 1977;98:427–433. 275 Neumann PE, Horoupian DS, Goldman JE, Hess MA. Cytoplasmic filaments of Crooke’s hyaline change belong to the cytokeratin class. An immunocytochemical and ultrastructural study. Am J Pathol 1984;116:214–222. 276a Asa SL, Kovacs K. Functional morphology of the human fetal pituitary. Pathol Annu 1984;19(1):275–315. 276b Pilardzic D, Kovacs K, Asa SL. Pituitary morphology in human anencephalic fetuses. Neuroendocrinology 1997;65:164–172. 277 Scheithauer BW, Kovacs K, Randall RV. The pituitary gland in untreated Addison’s disease. A histologic and immunocytologic study of 18 adenohypophyses. Arch Pathol Lab Med 1983;107:484–487. 278 Siperstein ER, Allison VF. Fine structures of the cells responsible for secretion of adrenocorticotropin in the adrenalectomized rat. Endocrinology 1965;76: 70–79. 279 Asa SL, Kovacs K, Hammer GD et al. Pituitary corticotroph hyperplasia in rats implanted with a medullary thyroid carcinoma cell line transfected with a corticotropin-releasing hormone complementary deoxyribonucleic acid expression vector. Endocrinology 1992;131:715–720. 280 Coates PJ, Doniach I, Hale AC, Rees LH. The distribution of immunoreactive a-melanocyte-stimulating hormone cells in the adult human pituitary gland. J Endocrinol 1986;111:335–342. 281 McNicol AM. A study of intermediate lobe differentiation in the human pituitary gland. J Pathol 1986;150:169–173. 282 Horvath SE, Asa SL, Kovacs K et al. Human pituitary corticotroph adenomas in vitro: morphologic and functional reponses to corticotropin-releasing hormone and cortisol. Neuroendocrinology 1990;51:241–248. 283 Horvath E, Lloyd RV, Kovacs K. Propylthiouracyl-induced hypothyroidism results in reversible transdifferentiation of somatotrophs into thyroidectomy cells: a morphologic study of the rat pituitary including immunoelectron microscopy. Lab Invest 1990;63:511–520. 284 Asa SL, Kovacs K, Bilbao JM. The pars tuberalis of the human pituitary: a histologic immunohistochemical, ultrastructural and immunoelectron microscopic analysis. Virchows Archiv (Pathol Anat) 1983;399:49–59.

Chapter 1 285 Horvath E, Kovacs K. Gonadotroph adenomas of the human pituitary: sexrelated fine structural dichotomy. A histologic, immunocytochemical and electron microscopic study of 30 tumors. Am J Pathol 1984;117:429–440. 286 Denef C. Paracrine interactions in the anterior pituitary. Clin Endocrinol Metab 1986;15:1–32. 287 Kovacs K, Horvath E. Gonadotrophs following removal of the ovaries: a fine structural study of human pituitary glands. Endokrinologie 1975;66:1–8. 288 Horvath E, Kovacs K, Penz G, Ezrin C. Origin, possible function and fate of “follicular cells” in the anterior lobe of the human pituitary. Am J Pathol 1974;77:199–212. 289 Girod C, Trouillas J, Dubois MP. Immunocytochemical localization of S-100 protein in stellate cells (folliculo-stellate cells) of the anterior lobe of the normal human pituitary. Cell Tissue Res 1985;241:505–511. 290 Höfler H, Walter GF, Denk H. Immunohistochemistry of folliculo-stellate cells in normal human adenohypophyses and in pituitary adenomas. Acta Neuropathol 1984;65:35–40. 291 Baes M, Allaerts W, Denef C. Evidence for functional communication between folliculo-stellate cells and hormone-secreting cells in perifused anterior pituitary aggregates. Endocrinology 1987;120:685–691. 292 Vankelecom A, Carmeliet P, Van Damme J et al. Production of interleukin-6 by folliculo-stellate cells of the anterior pituitary gland in a histiotypic cell aggregate culture system. Neuroendocrinology 1989;49:102–106. 293 Ferrara N, Schweigerer L, Neufeld G et al. Pituitary follicular cells produce basic fibroblast growth factor. Proc Natl Acad Sci USA 1987;84: 5773–5777. 294 Coates PJ, Doniach I. Development of folliculo-stellate cells in the human pituitary. Acta Endocrinol 1988;119:16–20. 295 Lauriola L, Cocchia D, Sentinelli S et al. Immunohistochemical detection of folliculo-stellate cells in human pituitary adenomas. Virchows Archiv (Cell Pathol) 1984;47:189–197. 296 Nishioka H, Llena JF, Hirano A. Immunohistochemical study of folliculostellate cells in pituitary lesions. Endocr Pathol 1991;2:155–160. 297 Kovacs K, Horvath E, Ryan N, Ezrin C. Null cell adenomas of the human pituitary. Virchows Archiv (Pathol Anat) 1980;387:165–174. 298 Asa SL, Cheng Z, Ramyar L et al. Human pituitary null cell adenomas and oncocytomas in vitro: effects of adenohypophysiotropic hormones and gonadal steroids on hormone secretion and tumor cell morphology. J Clin Endocrinol Metab 1992;74:1128–1134. 299 Yamada S, Asa SL, Kovacs K et al. Analysis of hormone secretion by clinically nonfunctioning human pituitary adenomas using the reverse hemolytic plaque assay. J Clin Endocrinol Metab 1989;68:73–80. 300 Jameson JL, Klibanski A, Black PMcL et al. Glycoprotein hormone genes are expressed in clinically nonfunctioning pituitary adenomas. J Clin Invest 1987;80:1472–1478. 301 Kontogeorgos G, Horvath E, Kovacs K et al. Null cell adenoma of the pituitary with features of plurihormonality and plurimorphous differentiation. Arch Pathol Lab Med 1991;115:61–64. 302 Kovacs K, Asa SL, Horvath E et al. Null cell adenomas of the pituitary: attempts to resolve their cytogenesis. In: Lechago J, Kameya T, eds. Endocrine Pathology Update. Philadelphia: Field and Wood, 1990;17–31. 303 Kovacs K, Horvath E, Bilbao JM. Oncocytes in the anterior lobe of the human pituitary gland. A light and electron microscopic study. Acta Neuropathol 1974;27:43–54. 304 Carafoli E. Mitochondrial pathology: an overview. Ann NY Acad Sci 1986; 488:1–18. 305 Horvath E, Kovacs K, Singer W et al. Acidophil stem cell adenoma of the human pituitary: clinicopathological analysis of 15 cases. Cancer 1981;47: 761–771. 306 Sano T, Kovacs K, Asa SL, Smyth HS. Immunoreactive luteinizing hormone in functioning corticotroph adenomas of the pituitary. Immunohistochemical and tissue culture studies of two cases. Virchows Archiv (Pathol Anat) 1990;471: 361–367. 307 Moriarty GC, Garner LL. Immunocytochemical studies of cells in the rat adenohypophysis containing both ACTH and FSH. Nature 1977;265:356–358. 308 May V, Wilber JF, U’Prichard DC, Childs GV. Persistence of immunoreactive TRH and GnRH in long-term primary anterior pituitary cultures. Peptides 1987;8:543–558. 309 Joubert (Bression) D, Benlot C, Lagoguey A et al. Normal and growth hormone (GH)-secreting adenomatous pituitaries release somatostatin and GH-releasing hormone. J Clin Endocrinol Metab 1989;68:572–577. 310 Pagesy P, Li JY, Rentier-Delrue F et al. Evidence of pre-prosomatostatin mRNA in human normal and tumoral anterior pituitary gland. Mol Endocrinol 1989;3:1289–1294.

Functional Anatomy of the Hypothalamic Pituitary Axis

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311 Duello TM, Nett TM, Farquhar MG. Fate of a gonadotropin releasing hormone agonist internalized by rat pituitary gonadotrophs. Endocrinology 1983;112:1–10. 312 Deschepper CF, Crumrine DA, Ganong WF. Evidence that the gonadotrophs are the likely site of production of angiotensin II in the anterior pituitary of the rat. Endocrinology 1986;119:36–43. 313 Major J, Ghatei MA, Bloom SR. Bombesin-like immunoreactivity in the pituitary gland. Experientia 1983;39:1158–1159. 314 Zaidi M, Abeyasekera G, MacIntyre I. Calcitonin gene-related peptide: endocrine distribution and characterization of circulating forms. J Endocrinol Invest 1989;12:699–704. 315 Rehfeld JF. Preprocholecystokinin processing in the normal human anterior pituitary. Proc Natl Acad Sci USA 1987;84:3019–3023. 316 Rehfeld JF, Lindholm J, Andersen BN et al. Pituitary tumors containing cholecystokinin. N Engl J Med 1987;316:1244–1247. 317 Vrontakis ME, Sano T, Kovacs K, Friesen HG. Presence of galanin-like immunoreactivity in nontumorous corticotrophs and corticotroph adenomas of the human pituitary. J Clin Endocrinol Metab 1990;70:747–751. 318 Vrontakis ME, Peden LM, Duckorth MC, Friesen HG. Isolation and characterization of a complementary DNA (galanin) clone from estrogeninduced pituitary tumor messenger RNA. J Biol Chem 1987;262: 16755–16758. 319 O’Halloran DJ, Jones PM, Steel JH et al. Effect of endocrine manipulation on anterior pituitary galanin in the rat. Endocrinology 1990;127: 467–475. 320 Kaplan LM. Gabriel SM, Koenig JI et al. Galanin is an estrogen-inducible, secretory product of the rat anterior pituitary. Proc Natl Acad Sci USA 1988;85:7408–7412. 321 Larson LI, Rehfeld JF. Pituitary gastrins occur in corticotrophs and melanotrophs. Science 1981;213:768–770. 322 Bardram L, Lindholm J, Rehfeld JF. Gastrin in pituitary tumors. Acta Endocrinol 1987;115:419–422. 323 Jones PM, Ghatei MA, Steel J et al. Evidence for neuropeptide-Y synthesis in the rat anterior pituitary and the influence of thyroid hormone status: comparison with vasoactive intestinal peptide, substance P, and neurotensin. Endocrinology 1989;125:334–341. 324 Goedert M, Lightman SL, Nagy IT et al. Neurotensin in the rat anterior pituitary gland. Nature 1982;298:163–165. 325 Morel G, Chayvialle JA, Kerdelhue B, Dubois PM. Ultrastructural evidence for endogenous substance P-like immunoreactivity in the rat pituitary gland. Neuroendocrinology 1982;35:86–92. 326 de Palatis LR, Fiorindo RP, Ho RH. Substance P immunoreactivity in the anterior pituitary gland of the guinea pig. Endocrinology 1982;110: 282–284. 327 Arnaout MA, Garthwaite TL, Martinson DR, Hagen TC. Vasoactive intestinal polypeptide is synthesized in anterior pituitary tissue. Endocrinology 1986;119: 2052–2057. 328 Steel JH, Gon G, O’Halloran DJ et al. Galanin and vasoactive intestinal polypeptide are colocalised with classical pituitary hormones and show plasticity of expression. Histochemistry 1989;93:183–189. 329 Lam SK, Reichlin S. Pituitary vasoactive intestinal peptide regulates prolactin secretion in the hypothyroid rat. Neuroendocrinology 1989;50: 524–528. 330 Webster J, Ham J, Bevan JS, Scanlon MF. Growth factors and pituitary tumors. Trends Endocrinol Metab 1989;1:95–98. 331 Ezzat S, Melmed S. The role of growth factors in the pituitary. J Endocrinol Invest 1990;13:691–698. 332 White BA, Bancroft FC. Epidermal growth factor and thyrotropin-releasing hormone interact synergistically with calcium to regulate prolactin mRNA levels. J Biol Chem 1983;258:4618–4622. 333 Murdoch GH, Potter E, Nicolaisen AK et al. Epidermal growth factor rapidly stimulates prolactin gene transcription. Nature 1982;300:192–194. 334 Polk DH, Ervin MG, Padbury JF et al. Epidermal growth factor acts as a corticotropin-releasing factor in chronically catheterized fetal lambs. J Clin Invest 1987;79:984–988. 335 Miyake A, Tasaka K, Otsuka S et al. Epidermal growth factor stimulates secretion of rat pituitary luteinizing hormone in vitro. Acta Endocrinol 1985;108:175–178. 336 Leriche VK, Asa SL, Ezzat S. Epidermal growth factor and its receptor (EGFR) in human pituitary adenomas: EGF-R correlates with tumor aggressiveness. J Clin Endocrinol Metab 1996;81:656–662. 337 Kasselberg AG, Orth DN, Gray ME, Stahlman MT. Immunocytochemical localization of human epidermal growth factor/urogastrone in several human tissues. J Histochem Cytochem 1985;33:315–322.

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338 Mueller SG, Kobrin MS, Paterson AJ, Kudlow JE. Transforming growth factor-a expression in the anterior pituitary gland: regulation by epidermal growth factor and phorbol ester in dispersed cells. Mol Endocrinol 1989;3:976–983. 339 Ezzat S, Walpola IA, Ramyar L et al. Membrane-anchored expression of transforming growth factor-≤ in human pituitary adenoma cells. J Clin Endocrinol Metab 1995;80:534–539. 340 Kobrin MS, Asa SL, Samoondar J, Kudlow JE. a-Transforming growth factor in the bovine anterior pituitary gland: secretion by dispersed cells and immunohistochemical localization. Endocrinology 1987;121:1412–1416. 341 Ramsdell JS. Transforming growth factor-a and -b are potent and effective inhibitors of GH4 pituitary tumor cell proliferation. Endocrinology 1991;128: 1981–1990. 342 McAndrew J, Paterson AJ, Asa SL et al. Targeting of transforming growth factor-a expression to pituitary lactotrophs in transgenic mice results in selective lactotroph proliferation and adenomas. Endocrinology 1995;136: 4479–4488. 343 Ying S-Y. Inhibins, activins, and follistatins: gonadal proteins modulating the secretion of follicle-stimulating hormone. Endocr Rev 1988;9:267–293. 344 Roberts V, Meunier H, Vaughan J et al. Production and regulation of inhibin subunits in pituitary gonadotropes. Endocrinology 1989;124:552–554. 345 Billestrup N, González-Manchón C, Potter E, Vale W. Inhibition of somatotroph growth and growth hormone biosynthesis by activin in vitro. Mol Endocrinol 1990;4:356–362. 346 Corrigan AZ, Bilezikjian LM, Carroll RS et al. Evidence for an autocrine role of activin B within rat anterior pituitary cultures. Endocrinology 1991;128:1682–1684. 347 Kogawa K, Nakamura T, Sugino K et al. Activin-binding protein is present in pituitary. Endocrinology 1991;128:1434–1440. 348 Penabad JL, Bashey HM, Asa SL et al. Decreased follistatin gene expression in gonadotroph adenomas. J Clin Endocrinol Metab 1996;81:3397–3403. 349 Gospodarowicz D, Ferrara N, Schweigener L, Neufeld G. Structural characterization and biological functions of fibroblast growth factor. Endocr Rev 1987;8:95–114. 350 Prysor-Jones RA, Silverlight JJ, Jenkins JS. Oestradiol, vasoactive intestinal peptide and fibroblast growth factor in the growth of human pituitary tumour cells in vitro. J Endocrinol 1989;120:171–177. 351 Elias KA, Weiner RI. Direct arterial vascularization of estrogen-induced prolactin-secreting anterior pituitary tumors. Proc Natl Acad Sci USA 1984;81:4549–4553. 352 Ezzat S, Smyth HS, Ramyar L, Asa SL. Heterogeneous in vivo and in vitro expression of basic fibroblast growth factor by human pituitary adenomas. J Clin Endocrinol Metab 1995;80:878–884. 353 Ezzat S, Zheng L, Zhu XF, Wu GE, Asa SL. Targeted expression of a human pituitary tumor-derived isoform of FGF receptor-4 recapitulates pituitary tumorigenesis. J Clin Invest 2002;109:69–78. 354 Stojlkovic SS, Merelli F, Iida T et al. Endothelin stimulation of cystolic calcium and gonadotropin secretion in anterior pituitary cells. Science 1990;248:1663–1666. 355 Matsumoto H, Suzuki N, Shiota K et al. Insulin-like growth factor-I stimulates endothelin-3 secretion from rat anterior pituitary cells in primary culture. Biochem Biophys Res Commun 1990;172:661–668.

356 Prager D, Melmed S, Fagin J. Feedback regulation of growth hormone gene expression by insulin-like growth factor I. In: LeRoith D, Raizada MK, eds. Molecular and Cellular Biology of Insulin-like Growth Factors and Their Receptors. New York: Plenum Press, 1989:57–71. 357 Yamashita S, Melmed S. Insulin-like growth factor I regulation of growth hormone gene transcription in primary rat pituitary cells. J Clin Invest 1987;79:449–452. 358 Berelowitz M, Szabo M, Frohman LA et al. Somatomedin-C mediates growth hormone negative feedback by effects on both the hypothalamus and the pituitary. Science 1982;212:1279. 359 Han VKM, Lund PK, Lee DC, D’Ercole AJ. Expression of somatomedin/ insulin-like growth factor messenger ribonucleic acids in the human fetus: identification, characterization, and tissue distribution. J Clin Endocrinol Metab 1998;66:422–429. 360 Yamashita S, Weiss M, Melmed S. Insulin-like growth factor I regulates GH secretion and mRNA levels in human pituitary tumor cells. J Clin Endocrinol Metab 1986;63:730–735. 361 Asa SL, Coschigano KT, Bellush L et al. Evidence for growth hormone (GH) autoregulation in pituitary somatotrophs in GH antagonist-transgenic and GH receptor-deficient mice. Am J Pathol 2000;156:1009–1015. 362 Asa SL, Kovacs K. Histological classification of pituitary disease. J Clin Endocrinol Metab 1983;12:567–596. 363 Shi T, Farrel MA, Kaufmann JCL. Fibrosarcoma Complicating irradiated pituitary adenoma. Surg Neurol 1984;22:277–283. 364 Del Pozo JM, Roda JE, Montoya JG et al. Intrasellar granuloma. Case Report. J Neurosurg 1980;53:717–719. 365 Asa SL, Bilbao JM, Kovacs K et al. Lymphocytic hypophysitis of pregnancy resulting in hypopituitarism. A distinct clinicopathologic entity. Ann Intern Med 1981;95:166–171. 366 Thodou E, Asa SL, Kontogeorgos G et al. Lymphocytic hypophysitis: Clinicopathologic findings. J Clin Endocrinol Metab 1995;80:2302–2311. 367 Sheehan HL, Stanfield JR. The pathogenesis of post-partum necrosis of the anterior lobe of the pituitary gland. Acta Endocrinol 1961;37:479–510. 368 Bergeron C, Kovacs K. Pituitary siderosis. A histologic and immunocytologic study. Arch Intern Med 1979;139:248-249. 369 Pfäffle RW, DiMattia GE, Parks JS et al. Mutation of the POU-specific domain of Pit-1 and hypopituitarism without pituitary hypoplasia. Science 1992;257:1118–1121. 370 Radovick S, Nations M, Du Y et al. A mutation in the POU-homeodomain of Pit-1 responsible for combined pituitary hormone deficiency. Science 1992;257:1115–1118. 371 Tatsumi K, Miyai K, Notomi T et al. Cretinism with a combined hormone deficiency caused by a mutation in the Pit-1 gene. Nature Gen 1992;1:56–58. 372 Li S, Crenshaw EB III, Rawson EJ et al. Dwarf locus mutants lacking three pituitary cell types result from mutations in the POU-domain gene pit-1. Nature 1990;347:528–533. 373 Daughaday WH, Trivedi B. Absence of serum growth hormone binding protein in patients with growth hormone receptor deficiency (Laron dwarfism). Proc Natl Acad Sci USA 1987;84:4636–4640. 374 Baumann G, Shaw MA, Winter RJ. Absence of the plasma growth hormonebinding protein in Laron-type dwarfism. J Clin Endocrinol Metab 1987;65: 814–816.

C h a p t e r

2 Adrenocorticotropin Mark A. Herman Joseph A. Majzoub

The anterior pituitary forms via invagination of the pharyngeal stomodeum in the region of contact with the diencephalon. By week 5 of human gestation, this invagination, termed Rathke’s diverticulum, has formed, but the downward migration of the diencephalic diverticulum, destined to be the neurophypophysis, has not yet commenced. It is at this time that adrenocorticotropin (ACTH) is first detectable by immunocytochemistry in the collection of cells within Rathke’s diverticulum which are furthest from contact with the diencephalon [1]. By 8 weeks of gestation, ACTH is detectable by radioimmunoassay of both fetal pituitary tissue and fetal blood [1]. The hypophyseal–portal vascular system forms between 8 and 14 weeks gestation, dating the earliest time after which hypothalamic corticotropin releasing factors may function to regulate fetal pituitary ACTH [1]. By 14 weeks gestation, release of ACTH from human fetal corticotrophs is highly responsive to exposure to corticotropin releasing hormone (CRH) in vitro [2]. The intensity of immunohistochemical ACTH staining in the anterior pituitary increases progressively from the mid-first through the end of the second trimester. In contrast, it is only after 21 weeks gestation that ACTH-positive cells begin to appear in the pars intermedia of the human fetal pituitary, defined as that region between Rathke’s cleft anteriorly and the neurohypophysis posteriorly. The ACTHcontaining cells in this region are epithelial-like, in contrast to the more angular, ovoid appearance of corticotrophs in the anterior pituitary [1].

those containing a-L-fucose and complex N-glyosylprotein, as well as terminal b-galactose. Corticotrophs constitute between 10 and 20% of the cell population of the anterior pituitary [3]. They are large cells with an ovoid or angular shape. They occur either singly or in clumps. They are most numerous in the midsagittal region of the pituitary (median wedge) but also occur in the lateral wings of the gland. Although the adult human pituitary does not contain an intermediate lobe, the junctional zone between the anterior and posterior lobes is known as the zona intermedia. This region, derived from the portion of Rathke’s diverticulum posterior to Rathke’s cleft, contains scattered cells immunopositive for ACTH. Some of these ACTHcontaining cells appear to extend into the posterior pituitary, a feature known as basophilic invasion [3]. These areas of apparent migration of corticotrophs from the zona intermedia into the posterior pituitary occur focally along the border between these two regions. In humans, in addition to the sellar pituitary, a pharyngeal pituitary exists which is located in either the sphenoid sinus or within the sphenoid bone. It consists of pituitarylike tissue approximately 2 to 5 mm by 0.2 mm in size. It is connected to the sellar gland by transsphenoidal vessels [4]. Only 1% to 2% of the cells in the pharyngeal pituitary contain immunoreactive ACTH, in contrast to approximately 14% of the cells in the sellar pituitary [4]. The pharyngeal pituitary is thought to arise as a rest of tissue resulting from the normal migration of cells from Rathke’s pouch to the sella turcica. There have been several reports of Cushing’s disease due to corticotroph adenomas arising in the pharyngeal pituitary [4,5].

Adult Anatomy

Molecular Embryology

Corticotrophs were initially identified by their basophilic staining. This has been subsequently found to be due to the presence of complex sugars in corticotrophs, principally

Tremendous progress has been made in recent years concerning the molecular mechanisms controlling pituitary organogenesis and pituitary cell-lineage specification in

CELLS OF ORIGIN

Fetal Anatomy

45

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animal models. A complex network of transcriptional events mediated by extrinsic and intrinsic signals has been implicated in the determination and a stereotypical spatiotemporal differentiation of the five trophic cell types of the mature anterior pituitary gland. Intrinsic Signals

RPX (for Rathke’s pouch homeobox), also known as HESX1 (for homeobox gene expression in embryonic stem cells) is the earliest known marker for the pituitary primordium [6]. HESX1/RPX is a member of the paired-like class of homeobox genes. Early HESX1/RPX expression occurs in the anterior midline endoderm and prechordal plate precursor, followed by expression in the cephalic neural plate [6]. Expression becomes progressively restricted anteriorly in the cephalic neural plate in a distribution consistent with tissue known by fate mapping studies to form the primordium of the anterior pituitary [7–10]. HESX1/RPX expression ultimately becomes restricted to Rathke’s pouch, and down regulation of HESX1/RPX in the pouch coincides with the differentiation of pituitary-specific cell types [6]. Embryonic mice lacking Rpx demonstrate variable abnormalities of hypothalamic and pituitary morphogenesis, reduced prosencephalon, anophthalmia or microophthalmia, and defective olfactory development. Neonatal mice have abnormalities in the corpus callosum, the anterior and hippocampal commisures, and the septum pellucidum [11]. These abnormalities are reminiscent of defects seen in a heterogeneous group of human disorders known as septooptic dysplasia (SOD). Deficits in SOD include optic nerve hypoplasia, abnormalities of the midline brain structures, and abnormalities of the hypothalamic-pituitary axis. Patients commonly present with endocrinopathies including hypoglycemia and adrenal crisis, which may signal growth hormone, ACTH, or thryoid-stimulating hormone deficiency [12]. Dattani and colleagues have identified two siblings with SOD who are homozygous for missense mutations within the HESX1 homeodomain which prevents it from binding target DNA [11]. The siblings identified with HESX1 mutations presented with hypoglycemia hours after birth and demonstrated hypopituitarism, substantiating a role for HESX1 in human pituitary development [11,13]. The corticotroph lineage appears to diverge relatively early from the other cell types of the anterior pituitary. LHX3, a LIM-type homeodomain protein, is essential for growth of Rathke’s pouch and determination of pituitary cell lineages [14]. In Lhx3-deficient mice, proopiomelanocortin (Pomc) was detected in a small cohort of cells at the ventral base of the pouch remnant which roughly corresponds to the position of the first presumptive corticotroph cells to differentiate in the wild-type pouch [14]. Although some pouch cells were able to differentiate and express Pomc, these cells failed to proliferate. An insufficient or non-functional corticotroph cell mass may have caused the hypoplastic adrenal cortices noted in these animals [14].

Ptx1 is a bicoid-related homeobox transcription factor identified based upon its ability to activate transcription of Pomc in the corticotroph derived AtT-20 cell line [15] and its ability to interact with the transactivation domain of Pit-1 [16]. It is expressed in the primordial Rathke’s pouch, oral epithelium, first branchial arch, the duodenum, and hindlimb [16]. Ptx1 is expressed in all mature pituitary cells, but is differentially expressed during pituitary development in different cell types [17]. In the mature pituitary, the highest levels of expression are found within corticotrophs [15]. In addition to activating transcription of Pomc, Ptx1 is required for sustained expression of Lhx3 [18], directly activates transcription of the common a-glycoprotein subunit (a-GSU) [16], synergizes with Pit-1 on the growth hormone and prolactin promoter [18], and synergizes with SF-1 on the promoter of the lutenizing hormone b (LH-b) gene [18]. Ptx2, also known as RIEG, is an additional bicoid-related homeobox gene 97% identical to Ptx1 [19]. Ptx2 is also differentially expressed in the pituitary and is excluded from corticotrophs [20], suggesting that some of the functions ascribed to Ptx1 may be performed by Ptx2. Expression of Pit-1, a POU domain transcription factor, is restricted to the anterior pituitary and required for the development of thyrotophs, lactotrophs, and somatotrophs [21]. Consistent with this, patients with mutations in PIT-1 have normal hypothalamic-pituitary-advanced (HPA) axis function [22]. Prophet of PIT-1 (PROP-1) is required for PIT-1 expression, and mutation of its gene is also associated with deficiencies in the development of thyrotrophs, lactotrophs, and somatotrophs in the Ames dwarf mouse and in humans. In a small number of patients with mutations in PROP1, modest impairment of ACTH secretion has been reported [23]. Developmentally essential, cell line-restricted factors have been identified for a number of different pituitary cell types. SF-1, an orphan nuclear receptor, is necessary for the development of gonadotrophs [24]. However, no essential corticotroph specific factor has yet been clearly identified. NeuroD, also known as Beta2, is a cell-restricted basic helixloop-helix (bHLH) transcription factor implicated in late neuronal differentiation [25] which has also been isolated as a cell-specific transcription factor of the insulin gene [26]. NeuroD is expressed in a number of tissues including pancreatic endocrine cells, the intestine, and brain including the mouse corticotroph AtT-20 cell line, but not the rat somatotroph pituitary cell line (GH3) [26]. Drouin and colleagues have recently demonstrated that NeuroD expression does appear to be restricted to corticotrophs in the mouse pituitary, and that NeuroD, in association with ubiquitous bHLH dimerization partners, specifically recognizes and activates transcription from the POMC promoter E box that confers transcriptional specificity of POMC to corticotrophs [27,28]. However, the necessity of this transcription factor for corticotroph specification or differentiation is still undetermined. Recently, a corticotroph-restricted transcription factor. TPIT, has been identified, which interacts with PTX1 and is required for POMC transcription, as discussed subse-

Chapter 2

quently. However, its role in corticotroph development is not known, and awaits further study. Because no instrinsic factor has yet been identified that is absolutely required for the development of corticotrophs in situ, it is possible that they arise elsewhere during fetal life and migrate into the anterior pituitary. One possible source could be neural crest, as this is the origin of POMC-producing melanocytes which migrate to the skin [29]. Extrinsic Signals

The oral ectoderm, from which Rathke’s pouch forms, and the neural ectoderm of the ventral diencephalon, from which the hypothalamus arises, are in direct contact at the time of the formation of Rathke’s pouch [30]. Classical explant experiments have indicated that inductive signals arising from mesenchyme and neural ectoderm adjacent to Rathke’s pouch are required for pituitary organogenesis and cell line specification [31]. The necessity for extrinsic signals for development of corticotrophs is supported by the demonstration that the expression of POMC in ectoderm explants requires coculture with mesoderm [32]. Direct evidence confirming the necessity of the ventral diencephalon for proper pituitary organogenesis has recently been obtained from examination of mice bearing null mutations in the Nkx2.1 homeobox gene, also known as T/EBP or TTF1 [33]. This gene is not expressed in the oral ectoderm or in the developing pituitary at any time during embryogenesis [34], but Nkx2.1-null mice demonstrate severe defects in the development of the diencephalon and also fail to develop a pituitary gland [33]. The nature of the extrinsic signals that promote pituitary organogenesis and cause cell-line specification is an area of active investigation. A number of secreted factors have been implicated in pituitary development and cell-line specification including bone morphogenic proteins (BMP4, BMP2), fibroblast growth factor 8 (FGF8), Sonic hedgehog (Shh) and Wnt5a [35–37]. A model of coordinate control of anterior pituitary progenitor cell identity, proliferation and differentiation imposed by FGF8 secreted from the dorsally located infundibulum and BMP2 from ventrally located mesenchyme has been proposed [37]. It has been suggested that corticotroph progenitors progress to a definitive corticotroph state only after escaping both FGF8 and BMP2 signaling [37]. While the precise signals and interactions required for pituitary organogenesis and corticotroph specification are incompletely understood, it is clear that signals from the mature hypothalamus like corticotropin releasing hormone (CRH) and vasopressin (AVP) are not required for corticotroph specification. The anterior pituitary appears to develop normally in a Crh-null mouse [38]. Furthermore, deletion of the class III POU transcription Brn-2 results in failed maturation of Avp-, Crh-, and oxytocin producing neurons of the hypothalamus and failed maturation of the posterior pituitary with no apparent defect in the maturation of any anterior pituitary cell type [39].

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Localization of Non-ACTH Peptides with Corticotrophs Many other neuropeptides have been found to be colocalized with ACTH within corticotrophs, although in most cases it is not clear whether this is due to synthesis or binding of the peptide within the cell. Neurophysin (but not vasopressin) colocalizes with ACTH in both normal and adenomatous corticotrophs [40]. Neurophysin immunocytochemical staining is especially prominent in corticotrophs in the zona intermedia which appear to project into the posterior pituitary. Chromogranin A has been described in the majority of corticotroph adenomas, although only a fraction of these patients have elevations of circulating plasma chromogranin A levels [41]. Galanin is present in all normal corticotrophs as well as in the majority of corticotroph adenomas which have been examined [42]. Galanin has also been found in those corticotrophs in the zona intermedia which appear to be migrating into the posterior pituitary. It has also been described in corticotrophs which have undergone Crooke’s hyalinization. Calpastatin, an inhibitor of the calcium-dependent cysteine proteases, calpain I, and calpain II, has been found in all ACTH-containing cells of the anterior pituitary, including those in the median wedge as well as those extending into the posterior pituitary [43]. Vasoactive intestinal peptide has been found in corticotroph adenomas, but only rarely in normal corticotrophs [44]. Normal corticotrophs contain small amounts of cytokeratin, whereas corticotrophs which have undergone Crooke’s hyalinization are markedly positive for this protein [45]. Corticotrophs appear to have few structural characteristics associated with neuronal cell types, for they are negative for neurofilament, vimentin, glial fibrillary acidic protein and desmon [45,46]. Likewise, corticotrophs in the zona intermedia, unlike those in the pars intermedia of rodents, appear not to be innervated by neurons, since they do not stain with any of these neuron-specific markers [47]. Similarly, synaptophysin, a 38 kD integral membrane glycoprotein found in presynaptic vesicles of neurons stains only weakly in corticotrophs [48]. These findings, together with a lack in humans of an anatomically discrete intermediate lobe with large numbers of a-MSH-containing cells, has led most investigators to conclude that there is no functional counterpart of the rodent pituitary intermediate lobe in humans.

Extra-pituitary Localization of ACTH and Related Peptides Although the vast majority of ACTH is synthesized in anterior pituitary corticotrophs, it is also expressed in several nonpituitary human tissues, both within and outside of the central nervous system. Within the central nervous system, ACTH and its related peptides are expressed to the greatest degree in cell bodies of the infundibular nucleus of the basal hypothalamus (analogous to the rodent arcuate nucleus) [49–51]. (See POMC Biosynthesis and Processing,

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pp. 52–53.) The cell bodies of these hypothalamic ACTH neurons are located at the base of the third ventricle, adjacent to the median eminence and pituitary stalk. These neurons project to limbic, diencephalic, mesencephalic, and amygdaloid sites [52]. Lesser amounts of ACTH and related peptides are found in substantia nigra, periventricular grey matter, and hippocampus [53]. Interestingly, POMC expression in the hypothalamus occurs in areas also known to express the orexigenic neuropeptides NPY and AGRP and project to many of the same hypothalamic targets [54,55]. The rodent arcuate nucleus prominently expresses the leptin receptor, implicating Pomc products in the regulation of appetite and energy homeostasis (see Melanocortin Receptor 4, p. 60) [56,57]. The brain areas containing ACTH also coincide with areas mediating stimulation analgesia, suggesting that expression of ACTH, or another product of the POMC gene (such as b-endorphin) in these sites may regulate pain perception [52]. Outside of the central nervous system, ACTH and other POMC gene products, including a-MSH and b-endorphin, are synthesized in a large number of human tissues, including in descending order of abundance, adrenal, testis, peripheral mononuclear cells, spleen, kidney, ovary, lung, thyroid, liver, colon, and duodenum [58,59]. In most of these tissues, POMC peptides are translated from truncated messenger RNAs lacking signal peptide sequences, suggesting that they cannot be secreted extracellularly [60,61], raising the question of their functional significance. However, adrenal and testis in addition express full length POMC mRNA, suggesting that these tissues may also secrete POMC-related peptides [58]. Recently, several additional cell types have been shown to produce POMC peptides including monocytes [62], astrocytes [63], gastrin-producing cells of the gastrointestinal tract [64], keratinocytes [65], skin melanocytes [66], and atrial myocytes [67]. GENE STRUCTURE ACTH is derived from a 266 amino acid precursor, proopiomelanocortin (POMC), so named because it encodes opioid, melanotropic, and corticotropic activities [68]. The human POMC gene is a single copy gene located on chromosome 2p23 [69]. It and the highly homologous opioid peptides, preproenkephalin A and preproenkephalin B (dynorphin), are all located on different chromosomes [70]. The human POMC gene is 8 kilobases (kb) long (Fig. 2.1). It consists of a promoter of at least 400 basepairs (bp) at the 5¢ end of the gene, followed by three exons, 86 (exon 1), 152 (exon 2), and 833 (exon 3) bp long, and two introns, 3708 (intron 1) and 2886 (intron 2) bp in length [69]. Exon 1 is not translated. Perhaps because of this, exon 1 of the human and other mammalian POMC genes are less than 50% identical. The initiator methionine is located 20 bp into exon 2, and is followed by a 26 amino acid hydrophobic signal peptide. Except for the signal peptide and 18 amino acids of the amino-terminal glycopeptide, the majority of

the POMC precursor is encoded by exon 3 [71]. Exon 2 is close to 90% identical between the POMC genes of humans and other mammals. Within exon 3 of POMC are located all known peptide products of the gene, including N-terminal glycopeptide, gamma-melanotropin (gammaMSH), joining peptide, a-MSH, ACTH, corticotropin-like intermediate lobe peptide (CLIP), b-lipotropin (b-LPH), b-MSH, and b-endorphin. Within exon 3, the regions encoding the N-terminal glycopeptide, a-MSH, ACTH, and b-endorphin, are greater than 95% identical between the human and other mammals [71]. In contrast, joining peptide, the region between the N-terminal glycopeptide and ACTH, is very poorly conserved among mammals [72], which has suggested to some workers that it does not encode a biologically important function [73].

POMC Gene Promoter Structure The promoter of the human POMC gene contains typical TATA and CAAT boxes 28 and 62 bp, respectively, upstream from the transcription start site. Using in vitro transcription [74] and transfection of the human POMC gene into heterologous cells [75], the POMC gene promoter has been shown to contain DNA elements which mediate increased transcription by cyclic AMP and decreased transcription by glucocorticoids. Although these DNA elements have not been precisely localized in the human POMC gene promoter, they are present with in the 700 bp 5¢ to the transcription start site [75]. The rat Pomc gene promoter has been studied more extensively. Drouin and coworkers, using DNA-mediated gene transfer into transgenic mice and tissue culture cells, found that the DNA sequences needed for corticotroph-specific expression and negative transcriptional regulation by glucocorticoid are contained within 543 bp of the transcription start site. These workers have reported the presence of a DNA sequence which mediates negative regulation by glucocorticoids (nGRE), located in the region of the CAAT box, which also binds nuclear proteins of the chicken ovalbumin upstream promoter (COUP) family. Using DNAase footprint and gel retardation analysis of the rat Pomc gene promoter, multiple synergistic DNA elements have been reported to be necessary for correct pituitaryspecific expression of the gene [76]. Recently, these workers have identified NeuroD, Ptx1, and Tpit as transcription factors which cooperate to cause corticotroph-specific expression of POMC [77].

POMC mRNA Transcription, Splicing, and Polyadenylation In addition to the major transcriptional initiation site present in corticotrophs, at least six other start sites have been found in several non-pituitary tissues (Fig. 2.1), including adrenal, thymus, and testes of the human [60] and rat [78]. These sites are all between 41 and 162 nucleotides downstream from the 5¢ and of exon 3. The mRNAs transcribed from

Chapter 2

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FIGURE 2.1. Structure of pro-opiomelanocortin (POMC) gene, messenger RNA (mRNA), and peptide products. (a) schematic diagram of gene (top), mRNA (middle) and POMC peptide (bottom). The gene consists of the promoter, 3 exons (thick bar, 1–3) separated by two exons (thin broken lines). Translated regions are stippled. The mRNA consists of 3 exons and the polyadenylate (AAAA) tail. The POMC precursor can be divided into 3 domains: I (N-terminal glycopeptide, joining peptide), II (adrenocorticotropic hormone; ACTH); and III (b-lipotropin; b-LPH). (b) Structure of individual products of POMC. Individual peptides are enclosed in different boxes depending on proteolytic cleavage specificities: = similar processing in pituitary and brain; = pituitary pattern of processing; = brain pattern of processing. = dibasic amino acids at proteolytic cleave sites. = glycosylation sites. = N-terminal acetylation sites. CLIP, corticotropin-like intermediate lobe peptide; a-MSH, a-melanocyte stimulating hormone.

these sites thus would be intronless, and the only truncated molecules that might be translated would be devoid of a signal peptide, and therefore could not be secreted [78]. Tissues containing these shorter forms of POMC mRNA, including adrenal, testis, spleen, kidney, ovary, lung, thyroid, and gastrointestinal tract, express ACTH, N-terminal glycopeptide1–61, and b-endorphin [58]. These truncated mRNAs are capable of being translated both in cell-free translation systems and in heterologous cells transfected with the appropriate fragment of the human POMC gene, although the peptide products are not secreted [61]. The significance of the expression of these truncated forms of POMC mRNA and their translated peptide products in human nonpituitary tissue is not clear.

A canonical polyadenylation signal is present in human POMC mRNA 23 bases upstream from the poly (A) addition site [79]. The length of the polyadenylate tail, attached to the 3¢ end of POMC mRNA and believed to play a role in translational efficiency of mRNA stability, is much longer in hypothalamic compared with pituitary cells in the rat [78], although this has not been studied in the human. GENE REGULATION In human anterior pituitary corticotrophs, POMC mRNA levels are increased by CRH and inhibited by glucocorticoids [80]. Similar results are seen in normal and adenoma-

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tous corticotrophs, although the glucocorticoid inhibition of POMC mRNA expression is less in the latter cells [80]. The same results are seen in rat anterior pituitary corticotrophs, where the opposing effects of CRH and glucocorticoids are due to their opposite influence on the transcription rate of the POMC gene [81]. Of interest, glucocorticoids have no effect on the POMC mRNA content of rat intermediate pituitary lobe corticotrophs [81]. CRH, acting via the type 1 CRH receptor [82] increases cAMP content in anterior pituitary corticotrophs [83]. CRH, vasopressin, and glucocotricoids all inhibit expression of CRH receptor 1 mRNA [84], which may limit the effect of these agents during the stress response, as discussed subsequently. The CRH-induced rise in cAMP is responsible for both the increase in POMC transcription and peptide synthesis as well as for the rise in intracellular calcium which results in ACTH secretion [85]. CRH mediates its stimulation of POMC transcription via the POMC CRH responsive element (PCRH-RE), which binds PCRH-RE binding protein [86]. The negative effect of glucocorticoids upon POMC gene transcription is thought to be mediated by a glucocorticoid–glucocorticoid receptor complex binding to cis-acting DNA sequences within the POMC promoter, although definitive evidence for this is lacking. The possibility exists that the glucocorticoid receptor complex does not bind directly to the POMC gene, but instead to another protein such as a positive transcription factor, and in this way mediates its negative effect on POMC gene expression [87]. Glucocorticoid stimulates, rather than inhibits, POMC gene expression [88] in the arcuate nucleus of the hypothalamus, the site of alphaMSH production. The significance of this is not clear, but could be involved in the inhibition of appetite by glucocorticoids in the postabsorptive state, when glucocorticoid levels are high. Recently, new insights into the development and regulation of the HPA axis have come from the discovery that leukemia-inhibitory factor (LIF) has an important role in these events [89]. LIF is a cytokine expressed in corticotrophs and folliculostellate cells beginning as early as 14 weeks gestation [90]. LIF stimulates transcription of POMC and expression of ACTH [90]. In many tissues, including pituitary, LIF expression is unregulated by inflammatory stimuli. The LIF receptor is a member of the class I cytokine receptor superfamily which heterodimerizes with gp130. In common with other family members, the LIF receptor signals through the Jak-STAT pathway, particularly utilizing Jak1 and STAT-3 [91]. As with other receptors coupled via these mediators, SOCS-3 inhibits POMC expression following its simulation by LIF [92]. Mice which overexpress a LIF transgene develop Cushing’s syndrome [93]. Their pituitary glands have corticotroph hyperplasia and multiple Rathke-like cysts lined by ciliated cells. Mice with targeted deletion of LIF have secondary adrenal insufficiency [94]. All of these data point towards LIF playing an important role in the regulation of ACTH secretion, perhaps most

importantly by immune and inflammatory stimuli [95]. In addition, the data suggest that LIF might be involved in the pathogenesis of Rathke-cleft tumors. Vasopressin potentiates the action of CRH on ACTH secretion, both in vitro [96] and in vivo [97]. Vasopressin’s effect is mediated by the vasopressin V1b (or V3) receptor and protein kinase C [98]. Despite this positive effect on ACTH secretion, vasopressin has been reported to decrease both basal and CRH-stimulated POMC mRNA levels in anterior pituitary cells [99]. b-adrenergic catecholamines, like CRH, also increase POMC mRNA levels in corticotrophs via a cAMP mechanism [100]. Insulin-induced hypoglycemia also causes an increase in POMC mRNA content in rat anterior pituitary corticotrophs [101,102], but whether this is secondary to an increase in hypothalamic CRH [101], vasopressin [103], or catecholamines [104] is not known. The inhibitory neurotransmitter GABA causes a decrease in POMC mRNA levels in intermediate, but not anterior pituitary corticotrophs [105]. POMC BIOSYNTHESIS AND PROCESSING The human POMC precursor has the potential to encode several overlapping peptides of biological importance (Fig. 2.1). Within the precursor, these peptides are separated from one another by two or more basic amino acids which serve as recognition sites for prohormone cleavage enzymes. In addition, POMC-derived peptides contain potential signals for amidation, glycosylation, acetylation and phosphorylation. Because the nomenclature of the various proteolytic products of POMC has been derived from both peptidemapping studies as well as molecular biological studies in which putative peptides had been predicted from inspection of nucleotide sequences [72], the terminology can be confusing. To avoid confusion, amino acid (aa) positions in this chapter are numbered as superscripts with reference to the 240 aa-long human POMC precursor, formed after removal of the 26 aa-long signal peptide. The 240 aa POMC precursor can be considered to be composed of three domains (Fig. 2.1). Domain I (aa 1–111), the N-terminal domain, encodes the 76 aa long Nterminal glycopeptide1–76 within its first 78 aa, and the 30 aa long C-terminal joining, or hinge, peptide ( JP79–108) within its last 33 aa. The middle Domain II (aa 112–152), 41 aa long, encodes the 39 aa ACTH112–150 peptide, which may be further processed to a-melanocyte stimulating hormone (MSH)112–124. The C-terminal Domain III, termed b-LPH153–240, is 88 aa long. It contains within it the 31 aa long b-endorphin210–240. Besides these peptides, several other products have been identified, although evidence for their existence and/or biological importance in man is not clear [106]. These include gamma1-MSH51–61, gamma2-MSH51–62, and gamma3-MSH51–76 in Domain I, corticotropin-like intermediate lobe peptide (CLIP)130–150 in Domain II, and gamma-LPH153–206 and b-MSH191–206 in Domain III (Fig. 2.1).

Chapter 2

Glycosylation of POMC Within the endoplasmic reticulum (ER), POMC undergoes initial glycosylation. Human POMC is glycosylated solely at two sites in the N-terminal glycopeptide1–76 of Domain I. Carbohydrate is added via an O-linked glycosylation to Thr45 and via an N-linked glycosylation to Asn65.

C-terminal Amidation of POMC Three human POMC products undergo C-terminal amidation [107]. These include N-terminal glycopeptide1–61, JP79–108, and -MSH112–124. In addition, these three products are also present in their Gly-extended forms, which may be incompletely-processed intermediates. C-terminal amidation is common among neuropeptides, and is usually essential for bioactivity [108]. This reaction is mediated by a bifunctional enzyme consisting of peptidylglycinea-amidating monooxygenese (PAM) and peptidly-ahydroxyglycine a-amidating lyase (PAL) activities, which transfer the amino group of a C-terminal Gly to the carboxyl group of the adjacent amino acid. Human PAM/ PAL exists in both a membrane-bound and free cytoplasmic form [109].

N-terminal Acetylation of POMC Two human POMC products undergo N-terminal acetylation. These are (as described below) a-MSH and bendorphin. In humans a-MSH exists predominantly in the nonacetylated form [110]. N-terminal acetylation of ACTH1–13-amide to form a-MSH results in increased melanotrophic activity [111] and decreased corticotrophic activity.

Proteolytic Processing of POMC POMC gives rise to several smaller, biologically active peptide products. These are generated by posttranslational cleavage of POMC by trypsin-like prohormone convertase endopeptidase enzymes which cleave the precursor on the C-terminal side of regions of two or more basic amino acid residues [112]. These basic amino acids are subsequently removed by a carboxypeptidase activity. Some POMC proteolytic products subsequently undergo amidation at their C-terminus or acetylation at their N-terminus, as described above. The posttranslational processing of POMC exhibits a remarkable degree of tissue-specificity, which recently has been postulated to be due to the differential distribution of processing enzymes in the various tissues which synthesize POMC (see below). Proteolytic Processing Enzymes

All proteolytic processing of human POMC occurs at either lys–arg or arg–arg residues (Fig. 2.1). Every lys–arg and arg– arg site within the human precursor is capable of being cleaved in vivo [106], whereas in the mouse and rat, additional arg–lys and lys–lys sequences at the N-termini of

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gamma1-MSH and b-MSH, respectively, appear to be utilized [106]. It is likely that the proteolytic digestion of human POMC at all sites is mediated by either of two structurally related endopeptidases, prohormone convertase 1 (PC1) [113] or prohormone convertase 2 (PC2) [113]. These enzymes, best studied in rodents thus far, are part of a seven member family of subtilisin/kexin-like mammalian proteinases [114], and are distributed specifically within endocrine cells and neurons. Both enzymes are capable of cleaving neuropeptide precursors, including POMC, proinsulin, and proglucagon, at dibasic sites, and each appears to manifest distinct preferences for different sites within the same precursor prohormone, with PC2 able to cleave at a wider selection of available dibasic sites than PC1 [115]. The tissue distributions of PC1 and PC2 mRNAs are distinctly different. PC1 is abundant in approximately 20% of anterior pituitary cells (presumably including corticotrophs), in all intermediate lobe pituitary cells (of the rodent), and in the supraoptic nucleus of the hypothalamus [116]. In contrast, PC2 is absent from the anterior pituitary, but is highly expressed in rodent pituitary intermediate lobe, multiple sites within the central nervous system, including cerebral cortex, hippocampus, and thalamus, and in pancreatic islet cells [116]. The differential tissue-specific distribution of these enzymes matches nicely with the known tissue-specific differences in POMC proteolytic processing (see below), suggesting that PC1 is responsible for the POMC cleavage products found in anterior pituitary corticotrophs, whereas PC2 cleaves POMC in pituitary intermediate lobe (of lower mammals) and in the brain. This suggestion is supported by studies of the differential processing of POMC by PC1 and PC2 [115,116]. Recently, a patient with severe childhood obesity and hyperproinsulinemia with postprandial hypoglycemia has been identified with compound heterozygous mutations in the PC1 gene [117]. The patient presented with multiple endocrine abnormalities including impaired glucose tolerance and postprandial hypoglycemia which has been attributed to the secretion of large amounts of proinsulin given its partial insulin-like action and long biological half-life. She also suffered from hypogonadotropic hypogonadism with primary amenorrhea, but otherwise normal development of secondary sexual characteristics. Ovulation was induced with exogenous gonadotropins, and the patient delivered healthy quadruplets. The pregnancy was complicated by gestational diabetes requiring insulin treatment. She also suffered from mild adrenal cortical insufficiency with complaints of fatigue and excessive daytime somnolence reversed by glucocorticoid administration. The adrenal cortical insufficiency was attributed to defective POMC processing with elevated levels of serum ACTH precursors confirming a role for PC1 in human POMC processing [117]. Following the generation of peptide products by prohormone convertase enzymes, the C-terminal basic amino acids are removed by carboxypeptidase activities. Carboxypeptidase E (Cpe) is required for the excision of paired

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dibasic residues of various peptide prohormone intermediates, including those derived from proinsulin and POMC [118]. The mutation in a strain of hyperproinsulinemic, late-onset obese mouse, the fat/fat mouse, has recently been mapped to the carboxypeptidase E gene [118]. A missense mutation has been identified in the Cpefat allele and these mice demonstrated a 20-fold decrease in Cpe enzymatic activity in pituitaries and isolated islets. The sorting of POMC into secretory granules is probably mediated by a signal “patch” [119], located within the tertiary structure of the molecule, which directs it to the granules. The presence of all POMC processed products in equimolar amounts within secretory granules suggests that this sorting precedes proteolytic cleavage of POMC. This suggestion is supported by data which demonstrates that initiation of proteolytic processing of POMC begins in the trans-Golgi system and continues in secretory vesicles, and is also consistent with data demonstrating localization of PC1 and PC2 within the TGN and/or dense core secretory granules [120]. Since POMC as well as PC1 and PC2 all contain signal peptides and are presumably colocalized throughout the endoplasmic reticulum, prohormone convertase activity must be inhibited in the endoplasmic reticulum and cis-Golgi regions. Proconvertase activity may be regulated by the local intracellular environment of these compartments and/or by control of catalytic activation of prohormone convertase. 7B2 is a small, hydrophobic acidic protein originally isolated from porcine and human anterior pituitary glands [121] that is widely distributed in neuroendocrine tissues [122] and found to associate specifically with PC2 [123]. The 27-kDa 7B2 precursor protein is cleaved to a 21-kDa protein and a small carboxy-terminal peptide (CT peptide) [124]. Interaction of 7B2, particularly the 21-kDa fragment, with proPC2 appears necessary for the generation of mature and active PC2 in the trans-Golgi region [125]. The 7B2 CT peptide is a nanomolar inhibitor of PC2 in vitro, but its role in vivo has not been defined. The role of 7B2 in activating proPC2 has been confirmed in vivo in 7B2-null mice [126]. 7B2-null mice are devoid of PC2 activity and deficiently process islet hormones and Pomc. The mice are hypoglycemic and demonstrate hyperproinsulinemia and hypoglucagonemia. These mice also demonstrate profound intermediate lobe ACTH hypersecretion with minimal conversion of this peptide to a-MSH, resulting in a severe Cushing’s syndrome that causes death by 9 weeks of age [126]. Curiously, PC2 null mice demonstrate similar islet cell dysfunction resulting in hypoglycemia, but do not abnormally produce a Cushingnoid syndrome [127]. This discrepancy suggests additional functional roles for 7B2, which are further suggested by the localization of 7B2 in regions of the brain lacking PC2 [128]. Recently, the suggestion that 7B2 may represent one member of a family of related convertase inhibitor proteins has been proposed with the identification of the protein proSAAS [129]. ProSAAS is a 26 kDa granin-like neuroen-

docrine peptide precursor isolated from rodents and humans with structural similarity to 7B2, including a proline-rich sequence in the first half of the molecule and a C-terminal peptide (SAAS CT peptide) following a dibasic cleavage sequence [129]. Overexpression of proSAAS in AtT-20 cells reduces the rate of Pomc processing and the SAAS CT peptide is a nanomolar competitive inhibitor of PC1, but not PC2 [129]. Tissue Specificity of POMC Processing

In human corticotrophs, POMC is processed predominantly into N-terminal glycopeptide1–76, joining peptide ( JP)79–108, ACTH112–150, and b-LPH153–240 (Fig. 2.1) [130]. Much smaller amounts of a-MSH112–124, CLIP130–150, bendorphin210–240, and a truncated form of N-terminal glycopeptide1–61, also known as “big” gamma-MSH, are also present [107]. There is no evidence for cleavage after arg50, and therefore no evidence for the presence of gamma1MSH51–61 in the human pituitary. JP79–108 exists as both a monomer and homodimer, most likely joined via disulfide bonding between the single Cys87 of two molecules [73]. Although JP79–108 has been postulated to stimulate adrenal androgen steroidogenesis [131], some studies do not support this hypothesis [73]. A function for N-terminal glycopeptide has also not been assigned, although one intriguing study reported that it is capable of stimulating aldosterone release from adrenal cells [130]. Whereas the production of distinct POMC peptide derivatives is clearly segregated between the anterior and intermediate lobes of the rodent, in the human, small, acetylated POMC peptide derivatives colocalize with larger desacetylated POMC peptides in corticotrophs of the anterior pituitary, suggesting that the strict dichotomy between corticotrope and melanotrope POMC processing observed in rodents and other species does not extend to human pituitaries [132]. Levels of desacetyl-a-MSH are elevated in pituitary corticotrophs [133] and plasma [134] of patients with Addison’s disease, Cushing’s disease, and Nelson’s syndrome. Desacetyla-MSH has approximately 75% of the melanotrophic activity as does a-MSH [111], whereas ACTH is only 5% as potent as a-MSH in this regard [111,135]. Because the serum levels of ACTH are 50- to 100-fold higher than levels of desacetyl-a-MSH in patients with Cushing’s disease, Addison’s disease, and Nelson’s syndrome, it is likely that the hyperpigmentation associated with these disorders is largely due to the melanotrophic effect of ACTH, and not MSH. Thus, in human anterior pituitary corticotrophs, the POMC precursor is predominantly cleaved at limited lys–arg sites into two peptides in Domain I (N-terminal glycopeptide1–76 and JP79–108), one peptide in Domain II (ACTH112–150) and one peptide in Domain III (b-LPH153–240) (see Fig. 2.1). As discussed above, human POMC is also expressed in several brain sites outside of the anterior pituitary, predominantly in the arcuate nucleus of the anterior hypothalamus. In these extra-pituitary locations, POMC is

Chapter 2

processed to a greater extent than in anterior pituitary. In brain, ACTH112–150 is cleaved to a-MSH112–124 and CLIP130–150 such that the amount of a-MSH relative to ACTH is 300-fold higher in hypothalamus, telencephalon, and mesencephalon than it is in anterior pituitary [136]. As in anterior pituitary, a-MSH is almost exclusively present in the desacetyl form [50]. Adding additional levels of complexity to the issue of tissue specificity of POMC processing are results from a dopamine D2 receptor (D2R) deficient mouse. Similar to dopaminergic tonic inhibitory control of prolactin expression in lactotrophs, POMC expression in the rodent intermediate lobe is under inhibitory dopaminergic control mediated via D2 receptors [137]. D2R-deficient mice demonstrate mild intermediate lobe hyperplasia accompanied by upregulation of both PC1 and PC2 [138]. These mice present with unexpectedly high levels of ACTH with corresponding adrenal hypertrophy and increases in corticosteroid secretion [138]. The altered prohormone convertase levels in these mice suggest the possibility of dynamic regulation of prohormone processing within specific tissues. HORMONE MEASUREMENT ACTH was one of the first substances to be measured by radioimmunoassay (RIA) [139]. In pioneering work using polyclonal antisera, Berson and Yalow described the measurement of ACTH and related peptides in normal persons and those with ectopic ACTH production by lung cancer [140]. These studies provided among the first data that ACTH was synthesized from larger precursors, which were termed “big” and “big–big” ACTH. The ACTH RIA can be performed on unextracted plasma, but suffers from a limit of sensitivity of approximately 25 pg/ml, and is therefore often unable to detect levels of plasma ACTH in the normal basal range. Radioimmunoassay remained the standard method for the measurement of plasma ACTH until the development of the immunoradiometric assay (IRMA). ACTH IRMAs employ two antibodies, one or both monoclonal, against ACTH. The solution-phase antibody is radiolabeled, and the solid-phase antibody is linked to a bead or other solid support. In general, the ACTH IRMA on unextracted plasma compares very favorably with RIA [141], being more sensitive, more reproducible, and more rapid [142]. Most IRMAs have lower limits of detection of 3–5 pg/ml and coefficients of variation of less than 10% up to 5000 pg/ml [141,142]. Depending on the antigenic specificity of the chosen antibodies, the ACTH IRMA may detect only intact ACTH [141], both ACTH and POMC precursor peptides [143,144], or only POMC precursors [145]. It is essential to know the sequence specificity of any IRMA in clinical use, for some, unlike most polyclonal RIAs, are incapable of detecting ACTH precursors which may be secreted by lung carcinomas [143]. Despite the wide availability of the ACTH IRMA, results from different laboratories are diffi-

Adrenocorticotropin

53

cult to compare because of lack of agreement on a suitable ACTH reference standard. An immunofluorometric assay (IFMA) for ACTH has also been developed [146]. Its sensitivity and accuracy are similar to those of the IRMA. Its principal advantage is its speed and the use of a nonradioactive label which is stable for over 1 year. Plasma assay of POMC products other than ACTH, including b-endorphin [147] and N-terminal glycopeptide, has been suggested as an adjunct in the evaluation of the hypothalamic-pituitary-adrenal (HPA) axis. In general, the levels of these hormones parallel that of ACTH in various HPA axis abnormalities [148]. However, except for use as a screening test for lung carcinoma associated with preferential secretion of N-terminal glycopeptide1–61 or proACTH1–150 [148], such tests are much less helpful than the ACTH IRMA because of the low concentration of other POMC peptide fragments compared with ACTH in most physiologic and pathologic settings. SECRETION OF ACTH: BIOCHEMISTRY AND PHARMACOLOGY Secretion of ACTH from the corticotrophs of the anterior pituitary is mediated by several factors (Fig. 2.2). CRH and vasopressin are the primary secretagogues for ACTH, although a number of other agents may also affect its release, and glucocorticoids are the major negative regulators of ACTH secretion. Once a ligand has bound to its receptor, release of ACTH from the corticortroph is mediated by second messengers through one of four signal transduction pathways, involving either protein kinase A, protein kinase C, glucocorticoids, or the Janus kinase/STAT system. (This

–

CRH

AVP

Catecholamines IL1β IL-6 TNFα

+

–

Cortisol

ACTH

Adrenal

+

+

LIF IL-6 IL1β TNFα

CRH IL1β IL-6 TNFα

FIGURE 2.2. Control of adrenocorticotropic hormone synthesis and secretion.

54

SECTION 1

Hypothalamic–Pituitary Function

last pathway, regulated by LIF, was described in a previous section, p. 50.) These pathways result in changes in the phosphorylation pattern of specific cellular proteins, and/ or in intracellular calcium levels, impacting on ACTH synthesis and release. Circulating ACTH then binds to receptors, primarily in the adrenal gland, leading to steroid biosynthesis.

Second Messenger Regulation of ACTH Secretion Protein Kinase A Pathway

CRH stimulates ACTH release via the cAMP-protein kinase A pathway. CRH stimulates adenylate cyclase activity, which then increases cAMP [83]. Forskolin, a direct stimulator of adenylate cyclase activity, and 8-bromo-cAMP, a cAMP analog, both markedly stimulate ACTH release, and increase CRH-stimulated ACTH release, but do not potentiate ACTH release from cells maximally stimulated by CRH [149]. The CRH-stimulated increase in cAMP activates cAMP-dependent protein kinase A [83]. Protein Kinase C Pathway

AVP V1b and V2 receptors are coupled to different second messenger systems. V1 receptors in the pituitary mobilize calcium through activation of phospholipase C, whereas V2 receptors activate adenylate cyclase, increasing cAMP [150]. AVP-stimulated ACTH release is mediated via the protein kinase C pathway [151]. Evidence that AVP acts through the protein kinase C pathway in the anterior pituitary includes the finding that AVP stimulates accumulation of inositol phosphates in rat anterior pituitary cells [152]. Phorbol esters, which activate protein kinase C directly by substituting for diacylglycerol and binding to protein kinase, induce ACTH release in rat pituitary cell cultures. Activation of protein kinase C appears more important in the sustained phase of AVP-stimulated ACTH release, not in the initial phase [153].

CRH CRH Stimulation of ACTH Secretion

Perfused human fetal pituitaries and cultured human fetal pituitary cells in culture secrete ACTH in response to CRH [2]. Rats have been used extensively in the study of ACTH secretion, as it is difficult to culture human corticotroph cells in vitro. In dispersed rat anterior pituitary cells, CRH stimulates a ninefold increase in ACTH release that is sustained for as long as the cells are exposed to CRH [83]. Even with maximal CRH stimulation, only 6% of cultured pituitary cells release ACTH [154]. CRH stimulates ACTH synthesis as well as release. In humans, the biphasic response may reflect secretion of a ready pool of ACTH, followed by later release of newly synthesized protein [155]. Exposing cells to CRH for a long period of time results in an increase in ACTH in the cell and in the medium. Thus, the sustained phase of ACTH

secretion may represent later release of newly synthesized ACTH peptide. Most of the CRH released into the hypophyseal blood is derived from the PVN [156]. Concentrations of oCRH that are similar to the concentrations of CRH found in rat portal plasma have been shown to increase secretion of ACTH in human fetal hemipituitaries in vitro [157]. The CRH content in the hypophyseal portal blood of anesthetized male rats is about 100 pM [158], which exceeds the in vitro threshold of 10–20 pM CRH to stimulate ACTH release [159]. In addition to stimulating ACTH expression and release, CRH can also directly stimulate secretion of glucocorticoids from the adrenal gland [160]. CRH Receptors

In human pituitaries, CRH has been shown to bind to sites in the anterior lobe [161]. The distribution of binding sites in human pituitary correlates with the distribution of corticotrophs [161]. CRH receptors in the anterior pituitary gland are low capacity, high affinity receptors, with a Kd for CRH binding of about 1 nM [162]. To date, two CRH receptor genes have been identified in humans and other mammals [163–165], with a third additional one being described in the catfish [166]. The type 1 receptor [163,165] is expressed predominantly in anterior pituitary corticotroph cells, whereas the type 2 receptor [164] is more widely distributed in the brain and periphery, particularly in cardiovascular tissue. The type 1 receptor binds and is activated by both CRH as well as the CRH-like peptide, urocortin [167]. This receptor mediates the actions of CRH at the corticoctroph. In addition, the type 1 receptor mediates fear and anxiety behaviors following stressors, even in CRHdeficient mice [168]. Mice with deletion of the CRH type 1 receptor gene show reduced fear and anxiety [169]. These data suggest that a CRH-related peptide, possibly urocortin or another unknown member of the CRH family, mediates fear responses via the CRH type 1 receptor. The CRH type 2 receptor binds urocortin with over 20-fold higher affinity compared to CRH. Its distribution, along with the hypotensive cardiovascular response to infused urocortin which is abolished in CRH type 2 receptor-deficient mice [170], suggests that the receptor may be involved in blood pressure control. This may underlie the hypotension observed during the CRH stimulation test. Recently, a nonpeptide, type 1-specific CRH antagonist has been developed which is orally active [171,172]. It should help to elucidate the role of CRH in ACTH regulation and other pathways.

AVP AVP Stimulation of ACTH Secretion

In rat anterior pituitary cells, AVP causes a twofold increase in ACTH release [173]. AVP elicits an initial rapid release of ACTH, observable within 5 seconds, reaching a maximum in less than 1 to 2 minutes, and lasting less than 3 to 6 minutes. This is followed by a second phase lasting

Chapter 2

for several hours. In humans, AVP infusion by itself has only a small effect on ACTH release. AVP is synthesized in the same parvocellular hypothalamic paraventricular nuclear neurons which express CRH, and appears to be coreleased with CRH at the median eminence into the portal hypophyseal system [174]. However, a substantial amount of AVP in the portal blood is released from projections from the supraoptic nuclei in the median eminence [156]. AVP from the posterior pituitary may also reach the anterior pituitary through portal vessels that connect the two [175]. This raises the possibility that increased vasopressin secretion from the posterior pituitary, in response to hyperosmolality, may also stimulate ACTH secretion. AVP Receptors

A single population of specific AVP receptors have been identified in rat anterior pituitaries, which are distinct from CRH receptors [176]. Most corticotrophs have AVP receptors, since 80% of the ACTH-secreting cells in the pituitary bind AVP [176]. The anterior pituitary AVP receptors are distinct from the V2 renal receptors and the V1 hepatic/pressor receptors [150]. This has led to the classification of hepatic/pressor receptors as V1a receptors and anterior pituitary receptors as V1b [150], or V3, receptors. V1a, V1b, and V2 receptors can be distinguished by their patterns of recognition of AVP analogues [177]. V1a binding sites in the rat anterior pituitary have a Kd of about 1 nM, and the minimal effective dose of AVP is 0.1 nM [173]. dDAVP (desmopressin), an AVP analog with V2 receptor affinity, has an insignificant effect on plasma ACTH levels, though it does increase, but is not additive to, CRHstimulated ACTH release [178]. During the past 6 years, the genes for all three vasopressin receptors (V1a, V2, and V1b) have been identified [179]. They are highly related members of the 7 transmembrane, G protein coupled, receptor family. As anticipated, V1b receptor mRNA is highly expressed in anterior pituitary corticotrophs, and is coupled to stimulation of POMC gene expression and ACTH secretion via the protein kinase C pathway [180,181].

Modulators of CRH and AVP Release CRH is the most important physiologic ACTH secretagogue [182]. Stressors, endogenous circadiam rhythms, and glucocorticoids influence CRH release [183]. In the rat, afferent inputs to the PVN may mediate the action of stressors by controlling the release of CRH [183]. Sources of neuronal afferents to the hypothalamus include the amygdala and hippocampus of the limbic system, and brainstem regions involved in autonomic functions [184]. In rats, acetylcholine, norepinephrine, angiotensin II, and possibly CRH itself, increase CRH concentrations in the hypophyseal portal plasma. On the other hand, AVP, b-endorphin, and GABA inhibit the ACTH response to stress [182,185–187].

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In rodents, central catecholamines stimulate ACTH release, and the effects of catecholamines on ACTH secretion appear to be mediated via secretion of CRH, and possibly AVP, into the hypophyseal portal circulation [188]. However, in humans, catecholamines have little direct effect on ACTH secretion from the pituitary. Peripheral catecholamines, increased by a variety of stresses, do not cross the blood–brain barrier to reach the hypothalamus, but do reach the pituitary, yet do not increase basal or CRHstimulated plasma ACTH levels. This suggests that the increased peripheral levels of epinephrine and norepinephrine generated during stress are probably not responsible for the increase in ACTH, and that catecholamines do not act directly on the pituitary to stimulate ACTH release [189]. In humans and other intact mammals, the impact of angiotensin II on basal or CRH-stimulated ACTH release is unclear. In humans, infusion of angiotensin II alone does not increase ACTH release [178]. However, in dispersed rat anterior pituitary cells, angiotensin II does stimulate release of ACTH [190]. Angiotensin II has a synergistic effect with CRH in stimulating ACTH release in humans in vivo [178]. Angiotensin II potentiates CRH-stimulate ACTH release from cultured anterior pituitary cells [191], although it is less effective than AVP, and potentiates the CRH-stimulated increase in cAMP. Angiotensin II and AVP do not potentiate each other’s effect on ACTH release, suggesting that they act via the same mechanism. Interleukin-1 beta stimulates ACTH release