General, Comparative and Clinical Endocrinology of the Adrenal Cortex

General, Comparative and Clinical Endocrinology of the Adrenal Cortex

VOLUME 2

I. CHESTER JONES

I.W. HENDERSON

Department of Zoology, University of Sheffield, Sheffield

Table of Contents

Cover image

Title page

Copyright

Contributors

Preface

Contents of Volume I

Contents of Volume III

Chapter 1: The Structure of the Mammalian Adrenal Cortex

Publisher Summary

1 Introduction

2 Methodology

3 General structure of the normal adult, Eutheria

4 Metatheria

5 General features of cell organelles of the mammalian adrenal cortex (Table X)

6 Cytochemistry and autoradiography at the ultrastructural level

7 The embryonic mammalian adrenal cortex: morphology and cytochemistry

8 The fetal or transient cortex and the X zone

9 Cytophysiology of the mammalian adrenal cortex

10 Stimulation in experimental hypoadrenocorticism

11 Problems associated with the structure of the mammalian adrenal cortex

12 Conclusion

Acknowledgements

Chapter 2: Steroidogenesis in the Zones of the Mammalian Adrenal Cortex

Publisher Summary

1. Introduction

2 Glucocorticoids

3 Mineralocorticoids: acute factors affecting secretion

4 Mineralocorticoids: chronic factors affecting secretion

5 The renin-angiotensin system

6 Functional aspects of adrenocortical zonation

7 Inner zone function

8 Metatheria

9 Adrenocortical sex steroids and gonadotropins

Acknowledgements

Chapter 3: Catabolism of the Adrenocortical Hormones

Publisher Summary

1 Introduction

2 Catabolic transformations

3 Catabolism of specific hormones

Chapter 4: The Adrenal Gland in Reptilia

Publisher Summary

1 Introduction

2 General morphology

3 Histology

4 Ultrastructure

5 Seasonal changes

6 The pituitary—adrenal axis

7 The hypothalamo-hypophysial axis

Part 2: PHYSIOLOGY

Publisher Summary

1 Adrenocortical control mechanisms

2 Corticomedullary relationships

3 Water and electrolyte balance

4 Protein, fat and carbohydrate metabolism

Chapter 5: The Adrenal Cortex of Amphibia

Publisher Summary

1 Introduction

2 Structure of the adrenal islets

3 Functions of adrenocortical secretions

Acknowledgements

Chapter 6: The Actions of Aldosterone

Publisher Summary

1 Introduction

2 Effects and actions of aldosterone

3 Mechanism of action of aldosterone

4 Conclusion

Chapter 7: Aldosterone Secretion and its Clinical Disorders

Publisher Summary

PART I. CONTROL OF ALDOSTERONE SECRETION

1 Controlling factors

2 Physiological variations in aldosterone secretion

PART II DISORDERS OF ALDOSTERONE SECRETION

Subject Index

Author Index

Copyright

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ISBN: 0-12-171502-7

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Contributors

P.J. Bentley,     Departments of Pharmacology, Opthalmology and Physiology, Mt. Sinai School of Medicine, The City University of New York, U.S.A.

I.P. Callard,     Biological Science Center, Boston University, Massachusetts, U.S.A.

G.V. Callard,     Harvard Medical School, Laboratory of Human Reproduction and Reproductive Biology, Boston, Massachusetts, U.S.A.

C. J. Edmonds,     MRC Clinical Research Centre, Harrow, Middlesex, England.

W. Hanke,     Zoologisches Institut, Universität, Karlsruhe, West Germany.

S. Idelman,     Laboratoire de Physiologie animale, Domaine Universitaire, Centre de Tri, Grenoble, France

C.J. Kenyon,     Department of Zoology, University of Sheffield, England.

D.E. Kime,     Department of Zoology, University of Sheffield, England.

B. Lofts,     Department of Zoology, University of Hong Kong.

W.N. Scott,     Departments of Pharmacology, Opthalmology and Physiology, Mt. Sinai School of Medicine, The City University of Mew York, U.S.A.

G.P. Vinson,     Department of Biochemistry and Chemistry, The Medical College of St. Bartholomews Hospital, London, England.

Preface

This, the second of three volumes devoted to the many facets of adrenocortical form and function, continues the multidisciplinary approach established in Volume 1. It is in many ways apt that publication of the 1976 Sir Henry Dale Lecture to the Society for Endocrinology (Chester Jones, I. (1976). J. Endocrinol. 71, 3–31) intercedes the appearance of Volumes 1 and 2 of this series. The lecture itself distills the attitudes that we have attempted to follow in the arrangement and subject matter. The mammalian adrenal cortex comes under scrutiny from structural, pathological, clinical, biochemical and histophysiological standpoints whilst the functional and structural characteristics of the adrenocortical homologue of two poikilothermic groups—the Reptilia and the Amphibia—are considered in more fundamental phylogenetic ways. Interspersed among these broad categories general biochemical and molecular biological discussions are given which put the other chapters into the general perspective that the adrenocortical homologue secretes materials that have an ubiquity of metabolism and mode of action in the Vertebrata.

We are very grateful to Mrs Nansi Chester Jones for compilation of the Subject Index and to Mrs Ruth C. Memmott, Mrs Elaine Bartlett and Mrs Jenny Noon for secretarial assistance. Many people very kindly supplied both published and new material and the detailsare noted in the appropriate legends. Whilst a complete list would be a duplcatiion of these, we should like particularly to mention: unpublished data on adrenalweights from Dr R. M. Laws for elephants, and Dr Idwal Rowlands for horses; Mr W. Mosley for the preparation of many figures and Mr D. Hollingworth for photography (Department of Zoology, University of Sheffield); Professor J. G. Phillips and Mr I. Carthy for original figures; Dr H. Saint Girons for the original slide of Sphenodon adrenalg land. In addition, the preparation of Chapter 1 was greatly helped by original illustrations acknowledged in the text, amongst whom may be noted: Drs R. S. Basch, Y. Y. Bertholet, V. H. Black, B. I. Bogart, O. von Deimling, H. Falk, M. J. Finegold, J. Frahm, W. W. Franke, J. Frühling, H. Fujita, M. Kloters, T. Ihara, E. Johannisson, A. L. Jones, J. A. Long, N. S. McNutt, P. A. Nickerson, G. G. Nussdorfer, J. A. G. Rhodin, M. Satre, J. H. Shelton, H. Sujihara, K. Unsicker and T. Zelander.

We record our thanks to the following for the use of previously published material : Acta Endocrinologica ; Adriatico Editrice, Bari ; Anatomische und entwicklungsgeschichtliche Monographien, Leipzig; Anatomical Record; American Journal of Anatomy; Beiträge zur pathologischen Anatomie und zur allgemeinen Pathologie; Bulletin of the Association of Anatomistes (Comptes Rendus de I’Association des Anatomistes); Bulletin of the Johns Hopkins Hospital; Cell Tissue Research; Endokrinologie; FEBS Letters; General and Comparative Endocrinology; Journal of Cell Biology; Journal of Experimental Molecular Pathology; Journal de Microscopie Société Français de Microscopie Electronique; Journal of Ultrastructural Research; Laboratory Investigation; Tissue and Cell; Zeitschrift für Zellforschung und mikroskopische Anatomie; The Carnegie Institute Washington Publications; Biochemical Endocrinology Series, Appleton-Century-Crofts; Kimpton, London; Plenum Publishing Corporation, New York.

I. Chester Jones and I.W. Henderson

Contents of Volume I

1. Structure and Nomenclature of Steroids. D. E. KIME and J. K. NORYMBERSKI

2. The Biosynthesis of Corticosteroids Throughout the Vertebrates. T. SANDOR, A. G. FAZEKAS and B. H. ROBINSON

3. Sub-mammalian Vertebrate Hypothalamic-Pituitary-Adrenal Interrelationships. C. BARKER JØRGENSEN

4. The Adrenal Cortex and the Alimentary Tract. R. J. LEVIN

5. The Adrenal Cortex of Birds. W. N. HOLMES and J. G. PHILLIPS

Contents of Volume III

1. Pisces

Part I. Structure. W. MOSLEY and I. CHESTER JONES

Part II. Physiology. I. W. HENDERSON and H. O. GARLAND

2. Adrenocortical Insufficiency (Addison’s Disease) and Overactivity (Cushing’s Syndrome). D. R. CULLEN, J. P. D. RECKLESS and E. H. McLAREN

3. The Regulation of the Pituitary-adrenal System in Mammals. I. VERMES and P. G. SMELIK

4. Adrenocortical Function in Relation to Mammalian Population Densities and Hierarchies. N. W. NOWELL

5. Pituitary-adrenal Hormones and their Roles in Adaptive Behaviour. B. BOHUS and D. de WIED

6. The Actions of Glucocorticoids. M. BEATO and D. DOENECKE

7. Adrenal-gonad Relationships. D. E. KIME, G. P. VINSON and P. W. MAJOR

8. Evolutionary Considerations. I CHESTER JONES, R. J. BALMENTand I. W. HENDERSON

1

The Structure of the Mammalian Adrenal Cortex

S. Idelman,     Laboratoire de Physiologie animale, Domaine Universitaire, B.P. 53, Centre de Tri, 38041, Grenoble Cedex, France

Publisher Summary

This chapter discusses the problems of adrenocortical structure using histochemical and ultrastructural techniques. In embryological development, adrenocortical cells arise from mesoderm, particularly from the columnar epithelial cells that line the coelom. Chromaffin cells have a separate origin, coming from neighboring paraganglion cells of the neural crest complex and thence migrating to lie adjacent to cortical cells. This intermingling is characteristic of most submammalian vertebrates though Elasmobranchii display complete separation. In Mammalia, the adult adrenal gland shows coalescence of chromaffin tissue and in Eutheria and Metatheria it lies centrally and is termed the medulla and eccentrically in the Prototheria. It is only over the past 10 years that the role of organelles in the biosynthesis of steroid hormones has begun to be delineated with correlated changes in fine structure. Changes occur in the smooth endoplasmic reticulum—the surface of which increases in area when the rate of steroidogenesis rises— and the mitochondrial cristae, which are usually tubular or vesicular in steroid secreting cells. Lamelliform cristae differentiate into tubular then vesicular cristae in most species studied. This is related to adrenocortical stimulation, for example, by ACTH. Thus, in hypophysectomized animals, the predominant tubular type is changed to vesicular by the administration of ACTH.

1 Introduction

2 Methodology

A. The time schedule of experiments

B. Stress

C. Fixation

D. Embedding

E. Ultrathin frozen sections

3 General structure of the normal adult, Eutheria

A. Gross anatomy

B. The vascular pattern within the adrenal

C. The lymphatic system

D. Nerves

E. The connective tissue framework

F. Adrenal weights

G. Micro-anatomy

H. Ultrastructure

4 Metatheria

5 General features of cell organelles

A. The endoplasmic reticulum

B. Mitochondria

C. Liposomes

D. Microbodies (peroxysomes) and lysosomes

E. Glycogen

6 Cytochemistry and autoradiography at the ultrastructural level

A. Cytochemistry

B. Autoradiography

7 The embryonic mammalian adrenal cortex: morphology and cytochemistry

A. Man

B. Rat adrenal cortex

C. The problem of light and dark cells

D. Monkey

E. Guinea pig

F. Cat

G. Armadillo

8 The fetal or transient cortex and the X zone

9 Cytophysiology of the mammalian adrenal cortex

A. Effects of inhibition of normal adrenocortical secretion

B. Effects of an inhibitor (cycloheximide) of protein synthesis

C. Effects of high sodium intake on the z. glomerulosa

D. Effects of inhibitors of steroidogenesis

E. Effects of stimulation of the adrenal cortex

F. Effects of stress or disease

G. Regeneration of the rat adrenal cortex

H. Inhibition and regulatory mechanisms

10 Stimulation in experimental hypoadrenocorticism

A. ACTH stimulation of dexamethasone-treated rats

B. ACTH action in hypophysectomized rats

C. Actions of cyclic-3’,5’ nucleotides on hypophysectomized rats

11 Problems associated with the structure of the mammalian adrenal cortex

A. Escalation and zonal theories

12 Conclusion

Acknowledgements

References

1 Introduction

The adrenal gland of the mammal is made up of two components, adrenocortical and chromaffin tissue. In embryological development, adrenocortical cells arise from mesoderm, particularly from the columnar epithelial cells which line the coelom. Chromaffin cells have a separate origin, coming from neighbouring paraganglion cells of the neural crest complex and thence migrating to lie adjacent to cortical cells (Chester Jones, 1976). This intermingling is characteristic of most sub-mammalian vertebrates though the Elasmobranchii display complete separation (Vol. 3, Chapter 1). In the Mammalia the adult adrenal gland shows coalescence of chromaffin tissue and in the Eutheria and Metatheria it lies centrally and is termed the medulla (Latin = the inmost part) and eccentrically in the Prototheria (Chester Jones, 1957). The relative morphological autonomy between mammalian cortex and medulla may not be so complete in terms of the inter-play of their secretions. A significant factor for these two tissues must relate to the vascular pattern of the adrenal gland. The vascularity favours the possibility of direct cortical action on medullary cells, promoting the methylation of noradrenaline to adrenaline (Coupland, 1953; Coupland and MacDougall, 1966; Wurtman and Axelrod, 1966). This is not necessarily true in lower vertebrates, (Wurtman, 1966; Wurtman et al., 1968), particularly the dogfish (Peyrin et al., 1969).

Medullary-cortical interrelationships are clearly important, though precise delineation is not yet forthcoming. For example, early work suggested that there was a pathway via hypothalamic stimulation and ACTH release (Long and Fry, 1945; Vogt, 1945; Long, 1947; Malmejac and Gross, 1950; Recant et al., 1950). Moreover, medullary hormones may act directly on cortical cell activity (Cooper et al., 1968). This is suggested by such experiments as unilateral electrical stimulation of the splanchnic nerve in the dog (Okinaka et al., 1960) and by the injection of adrenaline into the hypophysectomized guinea pig (Schweizer, 1953). Two actions are implied, one by increasing the vascularity of the gland and the second by adrenaline bathing the adrenocortical cells directly, or via centrifugal vascular loops from medulla to cortex (see Harrison, 1957; Harrison and Hoey, 1960). On the other hand, the medulla may not be necessary for adequate corticosterone secretion in the rat (Péron and McCarthy, 1966; Brudieux, 1971).

Arnold (1866) named the three main zones of the adult adrenal cortex which are discerned in histological preparations, the zona glomerulosa, the zona fasciculata and the zona reticularis (Chester Jones, 1957). A great deal of endocrinological work has been done with the laboratory rat so that it is appropriate to show the histological appearance of the gland in Fig. 1a,b. It encapsulates pictorially many of the problems explored over the years, such as the consequences of hypophysectomy and the different activities of the zones. Following a suggestion of Swann (1940), Deane and Greep (1946) developed the hypothesis that the three zones are morphologically distinct with separate secretions. This was termed the zonal theory by Chester Jones (1948) to distinguish it from the cell migration theory (Gottschau, 1883; see Chester Jones, 1957). The question of the secretions of the different cortical zones and their control is considered by Vinson and Kenyon (Chapter 2). It is generally agreed that the outer zone secretes aldosterone (a mineralocorticoid) which is associated with the regulation of electrolytes (Chapter 6). The glucocorticoids, principally Cortisol and corticosterone, are formed by the zona fasciculata and perhaps, in part, by the zona reticularis, and they are involved in protein and carbyhydrate metabolism (Vol. 3, Chapter 6). The adrenal cortex may normally secrete sex steroids (Chapter 2) as well as in disease (Sandor et al., 1976) and the zona reticularis may be responsible (Vaccarezza, 1945; Blackman, 1946; Race and Wu, 1961, for the whale; Ofstad et al., 1961, for man) though the zona fasciculata may also be implicated (Griffiths et al., 1963; Baillie et al., 1966; Chapter 2).

Fig. 1 (a) Transverse section of part of the adrenal gland of normal male Lister Hooden Rat aged 24 weeks. Capsule (CAP); zona glomerulosa (ZG); zona fasciculata (ZF); zona reticularis (ZR); medulla (MED). Bouin fixation, H and E stain. (× 120). (b) As for Figure la except hypophysectomized for 8 weeks. Note persistence of zona glomerulosa and degeneration of other zones. Bouin fixation, H and E stain. (× 120).

The zonation of the mammalian adrenal cortex is an intriguing and unexplained phenomenon. To touch on only one aspect, the zona glomerulosa may not be fully differentiated, having an unrepressed 18-hydroxylase system and that ACTH is able to convert its cells into the fasciculata type. This chapter is primarily concerned with the elucidation of problems of adrenocortical structure using histochemical and ultrastructural techniques.

2 Methodology

A THE TIME SCHEDULE OF EXPERIMENTS

There is a circadian rhythm in the secretions of the hypothalamo-hypophysial adrenal axis (Ganong, 1963; Critchlow, 1963; Critchlow et al., 1963). It follows, therefore, that comparable results can only be obtained by examination of animals killed at the same time of day. The daily rhythm means that the amounts of adrenocortical hormones vary throughout the day. Nadirs occur in man for Cortisol and ACTH from noon to 18.00 hours (Orth et al., 1967; Felbert and Aubert, 1968); rats, which are nocturnal, the lower secretory rates for corticosterone are from 08.00 hours to 16.00 hours and from 04.00 hours to 08.00 hours for CRF activity (Retiene et al., 1968; David-Nelson and Brodish, 1969; Hiroshige and Sakakura, 1971); for rabbits, corticosterone from 22.00 hours to 04.00 hours and Cortisol from 08.00 hours to 14.00 hours (Roussel et al., 1973); dogs from 16.00 hours to 20.00 hours (Digiesi et al., 1972). These considerations must be borne in mind when protocols for experimental design are produced.

B STRESS

Animals from which endocrine organs are to be obtained for structural examination must be killed without stress. This means that chloroform ether or barbiturates must be avoided (Carney and Walker, 1973; Pellet et al., 1970) and for this purpose a cervical blow is used. Prior to this, animal husbandry must be good so that abnormal noise and heat, for example, do not impinge. Inevitably, when glands must be fixed by perfusion, an anaesthetic is used and this is generally a barbiturate (e.g. Nembutal in rats at 60 mg/kg body wt).

C FIXATION

Good, quick fixation is required especially for the adrenal where cytolysis may supervene particularly rapidly. Many glands contain much lipid and the fixative used must take account of this. Histochemistry requires the use of frozen sections (by cryostat etc.) without fixation for enzyme studies and after slight fixation in formaldehyde or glutaraldehyde for lipids. For ultrastructure, the methods of Moses et al. (1969) and Frühling et al. (1969) are recommended. Thus:

1. 2% osmium tetroxide in a phosphate or barbital buffer at room temperature (Moses et al., 1969), for 12–24 hours

a. glutaraldehyde 2.5%; paraformaldehyde 2%; digitonin 0.2%; CaCl2 0.5% in 0.1 M cacodylate buffer at pH 7.2 for 2–21 hours

b. washing in cacodylate buffer 0.5 M with 0.05% CaCl2, three times for 10 min

2. osmium tetroxide 1% and CaCl20.05 M in cacodylate buffer 0.1 M for 1 or 2 h (Frühling et al., 1969; following Karnovsky, 1965; Scallen and Dietert, 1969); instead of immersion, glands may be perfused with glutaraldehyde; in some cases followed by osmium tetroxide (Christensen, 1965; Rhodin, 1971; Pearse, 1972).

D EMBEDDING

The most useful mixture for embedding adrenal tissue is Epon. Vestopal requires previous dehydration with acetone dissolving the lipids more completely than ethanol. Mollenhauer (1964) suggests a mixture of Epon and Araldite. With Epon, 90%, absolute ethanol is omitted and 70% alcohol is used as the final dehydrating agent. The mixture comprises equal volumes of 70% ethanol and Epon, twice each for 30 min (Idelman, 1964a,b). This gives about 90% retention of cholesterol (Moses et al., 1969; Frühling et al., 1969).

E ULTRATHIN FROZEN SECTIONS

Autoradiography of steroid ligands which may diffuse and are soluble in alcohol requires unfixed and unembedded tissue, and a cryoultramicrotome is necessary. Whilst the method of Bernhard and Leduc (1967) is useful (Idelman, 1972), it is better to obtain dry ultrathin sections as recommended by Stumpf and Roth (1966), Stumpf (1969) and Werner and Neumann (1972) without Dimethylsulphoxide, gelatine or water. Adrenal glands so far have not given satisfactory results (see Christensen, 1971).

3 General structure of the normal adult, Eutheria

A GROSS ANATOMY (Figs 2–4)

Reviews are to be found in Bachmann (1954), Chester Jones (1957), Deane (1962) among many others. The adrenal glands of the eutherian mammal are paired structures lying in the region of the anterior pole of the kidneys; though they vary in the closeness of their juxtaposition to them and to the main abdominal blood vessels. The glands are surrounded by both white and brown fat and are contained within the fascia of the kidneys. The glands take various shapes, spheroid, oval, elliptical, cylindrical or rod-like (Hartman and Brownell, 1949). In the dog, the right adrenal has the shape of a conch harpoon, the left, that of a dumbbell (Baker, 1937); in the rat the adrenals are beanshaped; in primates they are wedgeshaped. In man the gland has a concave lower surface which is applied closely to the cephalad end of the kidney.

Fig. 2 Ventral view of a dissection of the female rat of the Wistar albino strain (Rattus rattus) showing the vascular system associated with the adrenal glands after latex injection (× 2). (Prepared by I. Carthy and J. G. Phillips).

The general plan of the blood supply to the adrenals is similar throughout the Eutheria, though the details vary even amongst individuals of the same species. Some studies of blood supply may be noted: in the dog (Flint, 1900), in the cat (Bennett and Kilham, 1940), in the rat and mouse (Gersh and Grollman, 1941), in the rabbit, rat and cat (Harrison, 1951), in the rat (Lever, 1952), in the rhesus monkey (Harrison and Asling, 1955) and in man (Merklin and Eger, 1960; Merklin, 1962). The rat (Fig. 2) and the guinea pig (Figs 3 and 4) represent the typical appearance.

Fig. 3 Ventral view of a dissection of the female guinea pig (Cavia porcellus) showing the vascular system associated with the adrenal glands after latex injection. The right adrenal gland is displaced anteriorly to demonstrate arterial supply (× 2). (Prepared by I. Carthy and J. G. Phillips.)

Fig. 4 Ventral view of a dissection of the female guinea pig (Cavia porcellus) showing the vascular system, after latex injection, associated with the left adrenal gland which is twisted to the right side away from the kidney (× 5). (Prepared by I. Carthy and J. G. Phillips.)

The glands may receive branches from all the main arteries which pass near them and drain into the local major veins. In the dog, Flint (1900) found that the dorsal and ventral surfaces of the anterior part were supplied by three to five branches of the arteria phrenica, and of the posterior part by two to four branches of the arteria abdominalis; in addition, the posterior dorsal surface received two to six branches of the arteria lumbalis and, more caudally, four to six branches of the arteria renalis. These numerous adrenal arteries run, without anastomosing, from the main vascular trunks to the surface of the gland where they lie in the loose external connective tissue whence their twigs anastomose to form an ill-defined plexus. The venous drainage in the dog is not typical of the eutheria for as many as four large trunks empty into the lumbar vein which passes through the hilus on the ventral surface of the gland, and into the renal and phrenic veins.

In man, the general pattern shows the major portion of the blood supply as derived from the inferior adrenal arteries. These spring from the renal artery, at any point along its course, from the junction of the renal artery and the aorta or occasionally the aorta itself (Merklin, 1962; see also Gérard, 1913; Anson et al., 1947; Merklin and Michels, 1958). A large adrenal area is supplied by small numerous arteries given off by the inferior arteries or its posterior branch as they course over the diaphragm and along the superior border of the adrenal gland. These paired inferior phrenic arteries arise, singly or by a common trunk, from either the aorta or coeliac trunk. Merklin (1962) points out that a superior adrenal artery, though described in textbooks, does not occur.

In general, there is only a single vein leaving each gland. The vein in man on the right side is short and enters the inferior vena cava; on the left it descends to the renal vein receiving, on its way, the inferior phrenic vein and capsular tributaries. In the rat (Gersh and Grollman, 1941; Harrison, 1951), the anterior part of the gland on each side is supplied by an artery which, arising from the aorta, divides into two branches, and after further sub-division ramifies on the surface of the gland without apparent anastomoses. The medial part of the gland is vascularized by a separate artery arising from the ventral aspect of the aorta. A single adrenal vein on each side runs either directly to the vena cava or to the renal vein. In the cat the blood supply of the adrenal comprises numerous small arteries arising from the aorta, both renal arteries, the adrenolumbar arteries, one ileo-lumbar artery, the phrenic arteries and the coeliac axis. The main venous channels open directly through several small apertures into the adrenolumbar vein which lies in a depression on the face of the gland (Bennett and Kilham, 1940). The rabbit adrenal is chiefly supplied by subdivisions of a branch of the adrenolumbar artery, together with subsidiary vessels from the aorta so that ultimately about ten arteries reach the gland at various points around its periphery. The single adrenal vein on the left side enters the renal vein, on the right, the inferior vena cava (Harrison, 1951).

B THE VASCULAR PATTERN WITHIN THE ADRENAL

Flint (1900) gave the classical description of blood vessels within the adrenal gland (Fig. 5). The adrenal arteries form a network in the capsule to give branches supplying the whole gland. These small branches may have one of three separate courses: (1) they may be confined to the capsule where they terminate in an irregular capillary network which is gathered into widely anastomosed venous channels; (2) they may penetrate into the connective-tissue septa and break up immediately into an extensive capillary plexus which is confined entirely to the cortex—arteriae corticis; (3) they may penetrate the cortex without branching and be distributed as a coarse capillary plexus either directly or more distantly in the medulla—arteriae medullae. The cortical vessels on entering the cortex break up into fairly straight capillaries. They embrace the balls of cells of the zona glomerulosa, forming anastomoses of the arcade type. At the level of the zona fasciculata, the capillaries become straighter and run radially towards the centre of the gland giving off branches with varying degrees of anastomosis, though there are few in the rat. In the zona reticularis, the vessels widen into plexiform and irregularly anastomosing blood spaces or sinuses. These in turn empty into the medullary sinusoids draining into the central adrenal vein (two in the dog). Similar patterns occur in other mammals and the mouse and rat have been thoroughly studied (Fig. 6; Gersh and Grollman, 1941; Pauly, 1957; Ezaki, 1958). It is probable that the majority of cortical blood flows into the medulla and this would allow corticosteroids to play a part in the methylation of catecholamines (Wurtman et al., 1972). However, in man, Lazorthes et al. (1959) demonstrated a venous return to the outer part of the gland comprising some short and some long loops (Fig. 7). From the top of a loop a vein can be seen occasionally leaving to rejoin the veins of the medulla. These loops provide a functional balance in addition to the classical cortical drainage. Dempster (1974) found two circulations in the adrenal of man and the dog. The cortical circulation consisted of the cortical arteries and their veins drained into large trunks at the cortico-medullary interface and then directly via large trunks into the central vein. The medullary circulation consisted of the medullary arteries of Flint (1900) and most of their venae comites enter directly into the radicles of the central vein. Merklin and Eger (1960) found that drainage of the human adrenal gland is accomplished through successive orders of venules and veins which begin in the zona reticularis or in the medulla with many anastomoses. There are longitudinal muscles in the walls of the larger veins and these have been seen in the gibbon, chimpanzee, orang-utan, elephant, hippopotamus, rhinoceros and kangaroo though without any known function (Bargman, 1933).

Fig. 5 General anatomical relations and gross blood supply of the adrenals in an adult dog (from Flint, 1900). This diagram represents the separate encapsulated adrenal gland of the Amniota—mammals, birds and reptiles.

Fig. 6 Blood vessels of a part of the cortex and adjacent medulla of the adrenal gland of an immature rat, four weeks old (× 200). Injected via the heart with India ink diluted with an equal volume of distilled water, at a pressure of about 2 m of water. Formalin-alcohol, then 95% alcohol, embedded in celloidin, serially sectioned at 80 μm. Lightly stained in haemotoxylin and mounted in balsam. Reconstructed from camera lucida drawings (× 200) (Gersh and Grollman, 1941).

Fig. 7 Vascularisation within the adrenal gland of man (from Lazorthes et al., 1959, re drawn by W. Mosley, Department of Zoology, University of Sheffield, 1976). CAP: outer connective tissue capsule; ZG, zona glomerulosa; ZF, zona fasciculata; ZR, zona reticularis; MED, medulla. (a) This part gives the classic picture. (b) Arteriovenous loop with a capillary bed. (c) A simple arterio-venous loop. (d) An arterio-venous loop with sinusoidal capillaries. It will be noted that in addition to the classical picture there is venous return to the periphery of the gland with both short and long loops. MV, central medullary vein; MA, medullary artery.

The capillaries of the adrenal cortex, as well as those of other endocrine glands, are of the sinusoid type (Rinehart and Farquhar, 1955; Ham and Leeson, 1963; Bloom and Fawcett, 1975), i.e. they are lined with both endothelial cells and histiocytes (fixed macrophages). The latter are encountered mainly in the deeper layers of the zonae fasciculata and reticularis and are also found in the embryonic rissue. They are able to store particles of exogenous matter such as silver nitrate (Luse, 1961), India ink, iron, protein, trypan blue or lithium carmine (Idelman et al., 1974) thereby demonstrating their basic physiological property.

C THE LYMPHATIC SYSTEM

The first comprehensive report on the lymphatics of the adrenal gland was made by Kumita (1909) who found numerous vessels. These are disposed throughout the capsule, cortex and medulla and drain into a lymphatic meshwork surrounding the central vein. This meshwork, in turn, empties into two lymphatic trunks accompanying the adrenal vein and terminating in regional lymph nodes. Sapin (1959) found a similar arrangement in man. Merklin (1966) is in disagreement. He found lymphatic vessels only in the capsule and in the adventitia of the central vein and its largest tributaries (Fig. 8). Merklin considered that the random injection of dye by Kumita and Sapin brought out small veins which they confused with lymphatics.

Fig. 8 A composite drawing of the lymphatic drainage of the adrenal gland in man based on observations of 68 post mortem specimens. The adrenal lymphatic vessels are shown in black. The capsular channels begin blindly at scattered points on the gland surface usually in close association with one of the capsular vessels which form a small but important part of the adrenal gland venous drainage system. There are no tributaries from the cortical or medullary parenchyma to these capsular lymphatic channels. The majority of these run medially to the thoracic duct without the intervention of a lymph node. Some channels run caudad towards the kidney or cephalad towards the diaphragm (Merklin, 1966).

D NERVES

According to Greep and Deane (1949a,b) there is no nervous supply to the adrenal cortex and, nerve fibres, whether myelinated or not, which enter the gland end in the medulla. Nevertheless, the ultrastructure shows synaptic contacts between nerve fibres and cortical epithelial cells in the hamster (Unsicker, 1969), guinea pigs and man (Garcia–Alvarez, 1970), rats and pigs (Unsicker, 1971).

E THE CONNECTIVE TISSUE FRAMEWORK

The connective tissue framework follows the vascular pattern and seems to derive from that of the capsule. The groups of cells in the zona glomerulosa are outlined with thick septa of connective tissue fibres. The trabeculae then extend at right angles from this zone to the medulla, demarcating the columns of cells on the zona fasciculata and form a dense meshwork in the zona reticularis, each cell often being enclosed in a basket work of reticular fibres (Bourne, 1949). The prominence of the framework varies in different species. It is very pronounced in the sheep and cow, less so in the rat, mouse and man.

F ADRENAL WEIGHTS (Tables I, II; Fig. 9)

Data are given in Bachmann (1954), Chester Jones (1957), Deane (1962) and Arvy (1963) amongst others and demonstrate the variations according to species, age, sex and physiological states (Table I). Rowlands’ unpublished data on the horse is given in Table II. Laws has much unpublished data on the elephant and Fig. 9 gives a sample. It appears that increases in weight with age occur and there are many variations with sex.

TABLE I

The weights of adrenal glands of some mammals, relative to body weight. The table gives only a general indication as gland weights vary according to age, sex and physiological state of the individual. Based on Deane (1962) with additions. All figures are rounded upwards to the first decimal point. Figures in parentheses give the number of specimens.

TABLE II

The body and combined adrenal gland weights of the horse in embryonic and early post-natal stages. Data kindly given by Idwal Rowlands, Department of Anatomy, Cambridge University. The adrenal glands were fixed in Bouin’s fluid and weighed from 70 % alcohol after removal of excess fluid

Ab = aborted. Abortion was deliberately produced by removal of the cervical seal; this is the mucous plug in the cervix during pregnancy in equids.

pp = post partum.

Fig. 9 adrenal weights in g/kg body wt; numbers in parentheses give the number of elephants used for each point (Mosley, 1976). This text-figure was constructed by W. Mosley from data kindly supplied by Dr R. M. Laws with acknowledgements also to the Wildlife Service Ltd. The elephants were cropped in August, 1966 at Tsavao National Park, Kenya. The age was determined from the lower jaw by the method of Laws et al. (1967). The body weight was estimated from the weight of the hind leg (Laws et al., 1967). The paired adrenal weight with adnexa removed is given. Data when only one adrenal was weighed are discarded. See Laws et al. (1975) for a general account.

The ratio between adrenal weight and body weight appears to be approximately the same amongst most mammals, about 0.15 × 10--3. Adrenal weights probably bear a more direct relationship to the surface area of the animal rather than to the body weight. The general formula S = Kw²/³ where S is the surface area, w the body weight and K a constant with a value of about 10 for many animals (Benedict, 1938). The guinea pig and the mongolian gerbil have relatively very large adrenal weights for their body size (Chester Jones, 1957; Nickerson, 1971).

G MICRO-ANATOMY

1 General

Arnold (1866) named the three concentric zones of the cortex, a pattern donated by the growth and organization of the arterial vessels: the zona glomerulosa, lying below the outer connective tissue capsule, as balls or loops of cells, then the zona fasciculata of radially orientated cords of cells which break up in the innermost area as a less organized tissue, the zona reticularis (general references Bachmann, 1954; Chester Jones, 1957). The cortex in three major zones also presents sub-divisions and Nicander (1952) gives:

zona glomerulosa

zona intermedia or transitional zone (Greep and Deane, 1947), or

sudanophobe zone (Reiss et al., 1936)

zona fasciculata with an outer and inner zone

zona reticularis

medullary connective tissue capsule (present in most but not all species examined).

The volume of the gland occupied by the various zones clearly depends on the species and its physiological state. A general idea is given for man: zona glomerulosa 15%, zona fasciculata 78.4%, zona reticularis 6.4% (Swinyard, 1940); for the rat: zona glomerulosa 38%, zona intermedia 3%, zona fasciculata 54%, zona reticularis 5% (Sand et al., 1972); for the ox: zona glomerulosa 20%, zona fasciculata 51%, zona reticularis 29% (Howes et al., 1960).

The cortex viewed with the light microscope can show a very clearly defined zona glomerulosa in such species as the dog, horse and sheep and moderately so in the monkey, hamster, rat and mouse. The zona reticularis may not be prominent in some species (e.g. adult mouse, Chester Jones, 1948; hamster, Holmes 1950) and very marked in others (e.g. pig and guinea pig). The zona intermedia is readily seen in glands rich in lipid (e.g. dog and rat) as it does not take Sudan stains (Fig. 10). This zone changes in size or disappears in keeping with the physiological state of the animal, related, in the main, to cortical growth or renewal of cells (Section 9).

Fig. 10 The reaction of the rat adrenal cortex to Sudan Black B which shows the lipid content. The sudanophobic zone (intermediate zone) is seen at the junction of the zona glomerulosa and zona fasciculata. The medulla does not give a lipid reaction (× 60).

Other zones may be present, two of the most salient being the fetal or transient cortex in man (Starkel and Wegrenoski, 1910) and the X zone in the mouse (Howard–Miller, 1927)—the identification as zona reticularis by Tamura (1926) and Masui and Tamura (1926) was in error. The literature is given in Lanman (1953), Baar (1954), Sucheston and Cannon (1968) and Black (1972). These zones are discussed in Chapters 2 and 3.

2 Histology

The glomerular cells, in ovoid groups, constitute a narrow band immediately inside the capsule. The nuclei vary in shape from that of a sausage to oval. Nuclei have one or two nucleoli. The cytoplasm contains a varying number of lipid droplets (liposomes) which lie between the nucleus and that side of the cell abutting on the capillary. There are numerous mitochondria with rod- or thread-like shapes. They are smaller than those found in other zones (0.6 to 1 μm in diameter). The Golgi apparatus is adjacent to the nucleus but usually has no particular orientation towards the nearest capillary. The amount of lipid in the zona glomerulosa varies in different species under normal conditions. It is generally found that the dog, rat and hedgehog are particularly sudanophilic (Chester Jones, 1957; Girod et al., 1969) and the horse, mouse, pig, rabbit and cat less so (Bennett, 1940; Chester Jones, 1949a, b; Nicander, 1952).

The zona fasciculata comprises polyhedral cells considerably larger than those of zona glomerulosa. The cells have round nuclei with one or two nucleoli and the cytoplasm is packed with lipid droplets. The mitochondria are oval to spherical, predominantly ellipsoidal, with the long axes orientated parallel to the columns of the zone. It appears that there are sex differences in that usually the females have larger mitochondria (Dhom et al., 1971; Mäusle and Fröhlke, 1971). Again, the nuclei of the zona fasciculata of the female are larger by about 18% than those of the male with much smaller differences in the zona reticularis (2%) (Malendowicz, 1974). The nomenclature spongiocytes arises because the lipid is dissolved by routine histological procedures leaving artefactual vacuoles. Lipid droplets are practically all of the same size in any one cell. Frequently they are larger and more numerous in the outer zona fasciculata and smaller and fewer in the inner zone. In the hedgehog the outer two-thirds of the zona fasciculata has no liposomes while they are numerous in the inner third. In the human adrenal cortex, the zona fasciculata is less sudanophilic than the zona glomerulosa (Elias and Pauly, 1956).

The zona intermedia, when it occurs, has frequent mitoses (Section 11). The Golgi apparatus appears, in the cat, as a cap attached to the nucleus sending branches between the neighbouring liposomes. This is a commonly found pattern (Greep and Deane, 1949a,b). This zone can be found, though less clearly than in the rat cortex, in the horse, cattle, sheep, pig, dog, cat, rabbit, guinea-pig, mouse and hamster (Nicander, 1952). It has been considered as a zone forming the inner part of the zona glomerulosa or the outer part of the zona fasciculata (Lever, 1954). The interpretation of the zone is of necessity bound up with considerations of the entity of the zonae glomerulosa and fasciculata and how they are maintained (Sections 9, 10, 11).

The zona reticularis presents different aspects in different species, but, on the whole, it is never as wide as the zona fasciculata. The anastomosing networks of reticular cell cords surround the large blood sinuses. The cells are smaller than those of the rest of the cortex with less lipid content. There appear to be several different types of cell which at the ultrastructural level gives a confused picture (Section 3 H 1(c)).

Dostoiewsky (1886) found two types of cells in the zona reticularis, one characterized by a high affinity for heavy metals (siderophilia). This dichroism (Bernard and Bigart, 1902) was found by several authors. Da Costa (1913) described two types of cell in the zona reticularis, light and dark. The dark (= compact) cells stain intensively with all the usual cytoplasmic dyes and give a black colour with iron haematoxylin. These are Dostoiewsky’s siderophilic cells. They are, for the most part, smaller than the light cells and have numerous mitochondria and abundant pigment granules. There is little lipid, which occurs in small droplets. The light (= clear) cells contain lipid, have few mitochondria, little pigment and a large nucleus. Functional correlations are difficult to make. Originally Da Costa (1913) thought that the dark cells, with their irregular outlines and low content of liposomes, were those that had extruded their secretory products to go on to a new phase of cyclic secretory activity. It would seem that this stage is reversible in that the same cell can become light again if the lipid is restored. There are many intermediate types and I propose the word amphichroism instead of dichroism.

Some suppose that the clear cells of the zona fasciculata in man constitute a storage zone of steroid precursors, held in reserve. Compact cells of the zona reticularis, then, provide daily requirements. After ACTH, clear cells may transform into compact cells to give increased output of corticosteroids (Symington, 1962; Griffiths et al., 1963). A further modification supposes that both clear and compact cells contribute to the production of steroid hormones by the resting glands (Symington, 1969). The zona reticularis may well be an actively secreting zone (Symington et al., 1955; Symington, 1969). Mitoses do occur in the zone and, according to some authors, more frequently than in the zona fasciculata (Baxter, 1946; Mitchell, 1948; Cater and Stack–Dunne, 1953; Carr, 1959; Bertholet and Idelman, 1972). Cell death may be seen equally in any zone (Rhodin, 1971). The lipid content of the zona reticularis varies considerably though it is not so great, by and large, as that of the zona fasciculata. When lipids occur (and the hedgehog shows many, Girod et al., 1969) the droplets may run together to give large globules. Pigments form the lipofuchsins. These are not degraded by lysosomal hydrolases and undergo nonenzymatic auto-oxidation (Pearse, 1972). They appear to increase concomitant with age (Chester Jones, 1950; Szabo et al., 1970).

3 Histochemistry

(a) Sudanophilia and liposomes (lipid droplets)

Much work was directed to the cortical lipid droplets though they are not seen in some mammals such as the hamster (Alpert, 1950; Knigge, 1954; Girod et al., 1969), some hibernators (Deane and Lyman, 1954) or some ruminants (Nicander, 1952). Attention was originally drawn to depletion in number and reduction in size of the lipid droplets, coupled with changes in the organelles of the cells as indicative of the state of secretory activity (Deane et al., 1948; see Section 9). The lipid itself is made up of a mixture of cholesterol and its esters (among which the sulphates represent an important pathway of biosynthesis, Baulieu, 1962), palmitic, stearic and oleic esters of glycerol, the steroid hormones themselves and precursors (Pearse, 1972). Each lipid droplet (liposome on the terminology of Hoerr, 1936) comprises a mixture of all these compounds (Frühling et al., 1969; Sand et al., 1972). The comparative amount of lipid (liposomes) in the adrenal cortex of guinea-pig, rat and ox has been shown by Frühling et al. (1973; see Tables III and IV). In the rat, as a rough guide, about 14% of the total weight of the cortex is made up of lipid, 4.6% being cholesterol, most of which, about 83%, is esterified. Most of the cholesterol and its esters are found in the droplets (liposomes, about 75%; Tables III and IV). The question remains whether hormones or their precursors are stored in the liposomes. Rhodin (1971) has proposed such hormonal accumulation on the basis of morphological observations depicting extracellular discharge in the rat cortex (Section 9 E 1(c)). The hypothesis is favoured by the detection of pregnenolone in the lipid layers of supernatants in homogenates of dog adrenal glands (Holzbauer et al., 1973) and by the apparent storage of DOC in liposomes after metyrapone, shown by autoradiography of thin sections of rat adrenal cortex (Magalhães et al., 1974). However, further investigation is needed to throw light on the role of liposomes in steroid biosynthesis.

TABLE III

Volumes occupied by liposomes in the adrenocortical zones of three species. Results are expressed as a percentage of the total volume of adrenocortical cells

(from Frühling et al., 1973)

TABLE IV

Distribution of the principal constituents of sub-cellular fractions. Calculations from a standard rat adrenal cortex weighing 13 mg. Fraction weights given in μg (Sand et al., 1972)

(b) Cholesterol

Cholesterol and its esters are readily demonstrated histochemically and the methods will be found in Pearse (1972). The idea that a battery of tests could demonstrate and be specific for, adrenocortical hormones has long since been abandoned. The adrenal cortex in most species examined has a high content of cholesterol, reduced by ACTH (Sayers et al., 1944, 1946). Free cholesterol and its sulphates lie basically in the sequence to lead to corticosteroids (Sandor et al., 1976 in Vol. 1, Chapter 2). It can be seen, histochemically, predominantly in the zona fasciculata in the rat and monkey (Macacus). There are many variations and season also influences the pattern (Sivashankar and Prasad, 1968). Cell fraction techniques have shown the distribution of cholesterol in the rat (Table V). Intravenous or intra-cardiac injections of ³H-cholesterol allows the intra-cellular localization of cholesterol (Moses et al., 1969; Frühling et al., 1969, 1970, 1974c). After 45 minutes, under their conditions (q.v.) the labelled tissue is mostly the outer zona fasciculata of the rat, 25% of the total cholesterol activity in the mitochondria, 20% in the microsomal fraction and 36.3% in the liposomes. After 24 hours this becomes 79% in the liposomes, 15.6% in the mitochondrial fraction and 1.8% in the microsomes (Table VI).

TABLE V

Distribution of cholesterol in cell fractions of the rat adrenal cortex

(from Sand et al., 1972)

TABLE VI

Distribution (%) of ³H-cholesterol, after intravenous injection, with cell fractions of the rat adrenal cortex. Fractions were obtained by differential centrifugation and corrections made for exchange of ³H-cholesterol between fractions during centrifugation

(from Frühling, 1975)

Biochemical data on human adrenocortical slices specify distribution of cholesterol in the two inner zones (Griffiths et al., 1963). Reticular tissue (compact cells according to the authors) contains not only more nitrogen and DNA but also less cholesterol than equivalent wet weights of zona fasciculata (clear cells). Yet, the amounts of free cholesterol are similar in both zones (0.5–0.6 mg/100 mg tissue) and esterified cholesterol is the greater part, being about 6.5 mg/100 mg for zona fasciculata and 4 mg/100 mg for zona reticularis (see Frühling and Pecheux, 1976).

(c) Ascorbic acid

There is a large amount of ascorbic acid in the adrenal cortex as such figures as 3.9 mg/g adrenal weight (rat), 1.4 mg (cat) and 1.2 mg (guinea pig) indicate. It may be necessary for corticosteroidogenesis (Lowenstein and Zwemer, 1946) but in what way is not known. Changes in ascorbic acid content have been used as an index of adrenocortical activity as it is reduced by ACTH, indeed at a faster rate than cholesterol (Giroud and Ratsimamanga, 1942; Sayers et al., 1945, 1946). Histochemical methods are not specific (Pearse, 1972) and biochemical methods are preferred.

(d) Enzymes

(i) Δ⁵--3β-hydroxysteroid dehydrogenase

This enzyme was found in adrenal homogenates by Samuels et al. (1951). It is more or less associated with 5–4 isomerase and is able to convert dehydroepiandrosterone (DHA) or pregnenolone into corresponding 4–3 ketosteroids, androstenedione or progesterone. Wattenberg (1958) applied the method at the histochemical level and with the modification of Levy et al. (1959) is widely used. Steroid-producing glands of many animals have been examined (see Baillie et al., 1966) and cells, when positive, are stained blue by the diformazan salt (Fig. 11). The distribution of the enzyme activity in the adrenal cortex varies with the species, age, sex and physiological state. It seems generally true that the outer zona fasciculata gives the strongest reaction (rat, Wattenburg, 1958; Freses et al., 1965; man, Cavallero and Chiappino, 1961; Dawson et al., 1961a). Biochemically, the light cells of the zona fasciculata in man have a higher activity than the dark cells of the zona reticularis (Grant, 1964). In the rat after hypophysectomy the activity remains in the zona glomerulosa but disappears in the inner zones which can then be restored by ACTH (Levy et al., 1959). The activity in the zona glomerulosa can be increased by measures which lead to increased aldosterone production such as restricted sodium intake or angiotensin injections (Marx and Deane, 1963).

Fig. 11 Δ⁵,3β-dehydrogenase activity in the rat adrenal gland using DHA as a substrate. The activity appears higher in the zona fasciculata and zona reticularis (a, × 25; a’, × 60).

The enzyme is mainly located in the endoplasmic reticulum (Dorfman and Ungar, 1965; Inano et al., 1969a). It also occurs in the mitochondrial fraction of rat adrenal homogenates giving some 31% of the total activity whereas 45% is found in the microsomal fraction (Basch and Finegold, 1971). As the enzyme has also been localized in the inner membrane fraction of mitochondria from rat testis (Sulimovici et al., 1973), the isolation in this fraction from cortical tissue by McCune et al. (1970) and Basch and Finegold (1971) may not simply be microsomal contamination.

The histochemical method for this enzyme is particularly useful. Apart from its obvious applications, it is valuable in detecting suspected cortical tissue when cortical and medullary tissue are intermingled. This occurs in the medulla of the Eutheria from time to time and it is a characteristic feature of most sub-mammalian vertebrates.

(ii) Diaphorases and other dehydrogenase activities

Dawson et al. (1961b) pointed out that the detection of NAD- and NADP-diaphorase activity, together with sudanophilia and Δ⁵--3β-dehydrogenase activity, are the three most valuable methods in the histochemical evaluation of adrenocortical function. Δ⁵--3β-dehydrogenase activity is linked with NAD (NADP) so that its histochemical demonstration requires an NAD-diaphorase to be present in the adrenal gland. The mouse (Allen, 1959), hamster (Gospodinov and Davidoff, 1971), rat and man (Davignon et al., 1960; Dawson et al., 1961b) have been examined. A reaction is given throughout the cortex but of course there are variations concomitant with age, sex and physiological status. Thus the reaction is weaker in the zona glomerulosa of the rat and stronger in that of the hamster compared with other zones. ACTH, as would be expected, increases the activity (NADH tetrazolium reductase). Usually there is some increase after metyrapone treatment though a decrease sometimes occurs as it does after Cortisol administration (Malendowicz, 1973).

One of the more interesting dehydrogenases is glucose-6-phosphate dehydrogenase (G-6-PD) which develops a stronger reaction in the adrenal cortex than in some other tissues (Table VII). This enzyme is connected with steroid biosynthesis, catalysing oxidation of G-6-PD which is coupled to NADP reduction. It generates NADPH which is involved in C-21 and C-11 hydroxylations (Sandor et al., 1976). Hence, inhibition of steroid biosynthesis by oestrogens or progesterone is related to G-6-PD (McKerns and Kaleita, 1960). The enzyme gives strong reactions in the rat zona fasciculata and zona reticularis and less so in the zona glomerulosa (Cohen, 1959). There is a strong reaction at the junction of the zonae glomerulosa-fasciculata in the rabbit (Jonadet et al., 1971). In the hamster the zona glomerulosa develops the strongest reaction compared with other zones.

TABLE VII

Glucose-6-phosphate dehydrogenase activities* in some tissues compared with those in the adrenal gland

*Expressed in Glock and McLean units, i.e. the activity which reduces 0.01 μmol of NADP per min at 25°C (Glock and McLean, 1954).

†From Kelly (1955).

ACTH regulates much of the activity of the adrenal cortex and, in turn, that of G-6-PD activity (Greenberg and Glick, 1960 in the rat; Studzinski et al., 1962 in man). One type of estimation for the normal rat adrenal cortex gives an arbitrary number of 109, increased by ACTH to 115 zona glomerulosa, 145 zona fasciculata, 108 zona reticularis (57 medulla) (Greenberg and Glick, 1960). Another enzyme, the 6-phosphogluconate dehydrogenase, is developed more weakly than G-6-PD but shows a similar distribution so that following the arbitrary numbers given above, the values are glomerular-fascicular zone, 46; zona fasciculata, 77; zona fasciculata-reticularis, 21; zona reticularis 51, with the medulla giving a weak response (11). ACTH increases the activity.

(iii) Sulphatases and β-glucuronidase activities

Sulphatase activity