Histoenzymology of the Endocrine Glands: International Series of Monographs in Pure and Applied Biology: Modern Trends in Physiological Sciences

HISTOENZYMOLOGY OF THE ENDOCRINE GLANDS

L. ARVY, D.M., D.Sci.

Histoenzymological Laboratory, Faculty of Medicine, Paris VI

Table of Contents

Cover image

Title page

Copyright

PREFACE

INTRODUCTION

Chapter I: DIENCEPHALIC ENDOCRINE GLANDS

Publisher Summary

A. Anatomical and Physiological Survey

B. Enzymic Activity

C. Discussion

IN THE INVERTEBRATES

A. Histophysiological Summary

B. Enzymology

IN THE VERTEBRATES

A. Anatomical and Physiological Survey

B. Enzymic Activity

THE CAUDAL SPINO-HYPOPHYSEAL COMPLEX OF FISH

A. Morphology

B. Histoenzymology

Chapter II: THE PARATHYROID AND THYROID GLANDS

Publisher Summary

Introduction

A. The Parathyroid Glands*

B. Parathyroid Enzymic Activity

A. The Thyroid Vesicles

B. The Clear Cells*

C. Thyroid Enzymic Activity

Chapter III: ENZYMIC ACTIVITY IN THE ISLETS OF LANGERHANS

Publisher Summary

Introduction

A The Histoenzymology of the Islets of Langerhans in Poikilotherms

DISCUSSION

B Histoenzymology of the Islets of Langerhans in Birds

C Histoenzymology of the Islets of Langerhans in Mammals

DISCUSSION

ENZYMES

Chapter IV: THE ADRENAL GLAND

Publisher Summary

Introduction

A The non-mammalian adrenal gland

B The Mammalian Adrenal Gland

CONCLUSION

Chapter V: ENZYMIC ACTIVITY OF THE GONADS

Publisher Summary

Introduction

THE GERMINAL CELLS

THE FEMALE GONADS

Introduction

A The Gonads of Fish

B The Amphibian Gonad

C The Gonads of Reptiles

D The Gonads of Birds

E The Gonads of Mammals

INDEX

OTHER TITLES IN THE SERIES

Copyright

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Original Copyright © 1963 Gauthier-Villars

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PREFACE

IT IS not excessive presumption that causes me to give an account of the histochemically detectable enzymic activity in the endocrine glands. I know my limitations and I am aware of the difficulties of this enterprise. However, it seems more and more clear that we are capable of understanding some of the links in the coordinating enzymic chains which lead to the biosynthesis of molecules endowed with hormonal activity. I have the conviction that it is time for histochemists to contribute to endocrinology in an attempt to superpose an enzymic architecture on the endocrine glands just as a hundred years of research has established a histological architecture. Considerable work is needed which will only be built up by numerous and varied contributions. At the moment, an isolated worker can only dream of a rough sketch of the distribution of the histochemically detectable enzymic activity in the endocrine glands, and especially he considers their modification during ontogenesis and their variation during physiopathological states.

Extent and Limit of the Subject

The endocrine glands can be defined as peculiar formations, branching from the origin on blood and lymphatic systems. Lacking ducts, they liberate the product they elaborate directly into the blood or lymph. Physiologists have not succeeded in characterising the endocrine role of certain structures lacking ducts, such as the spleen, thymus, lymphatic nodes, Fabricius pouch, etc. Further biochemists have not succeeded in isolating a specific active principle. Therefore, I am only dealing with histochemically detectable enzymic activity in glands associated with the vascular system whose endocrine nature is proved, that is to say, the pineal, hypothalamo-neurohypophysis, adenohypophysis*, thyroid and parathyroid, islets of Langerhans, adrenal and gonads. The embryological origins of the endocrine glands are varied. Some are ectodermal, others mesodermal. Some have a relatively simple and stable structure such as the parathyroid, others such as the pars distalis of the adenohypophysis have a complex structure so variable in different physiological states that endocrinologists are still arguing about the number of cell types they contain. The hormonal principles elaborated by these various glands are fundamentally different.

*= pars distalis + pars intermedia + pars tuberalis.

INTRODUCTION

LUCIEN LISON

IT IS particularly pleasing to us to be able to present, as our first title in Monographs on Histochemistry and Dynamic Morphology, the present work by Mlle L. Arvy. For many years Mlle Arvy has specialised in histoenzymology and has become one of the undisputed masters of this branch of histochemistry. Progress in histoenzymology is particularly rapid and periodical reviews are necessary so that the exact significance of the advances can be appreciated. Mlle Arvy, in addition to considerable activity in original scientific research, attacked this task with rare good humour. Several years ago Mlle Arvy published, under the modest title Present Techniques in Histoenzymology, a very remarkable monograph, whose scope passed most of the limits of the usual compilations of histochemical technique. The reader will find again in the present work, devoted to histoenzymology of the endocrine glands, breadth of documentation, clarity of thought and firmness of style that all had admired in the first work. As previously, Mlle Arvy is not content to relate faithfully the histoenzymological facts. She has sought, with ability, the significance and integrated it with our knowledge of the histophysiology of the endocrine glands, which, throughout this work, is recalled and explained with extraordinary precision and clarity.

CHAPTER I

DIENCEPHALIC ENDOCRINE GLANDS

Publisher Summary

The numerous investigations over the past decades have led to a satisfactory analysis of the anatomical and physiological connections of the hypothalamic-hypophyseal complex. Until these past years, the differences in coloration, observable with more or less difficulty, have only been a clue to the different staining affinities of the different hormonal products synthesized by these cells. Demonstration of the enzymic activities allows one to study directly the mechanisms that result in the synthesis of various hormones. The presence of an active peptidase in the pineal gland suggests that the peptides are elaborated inside the pineal cells. The study of all the reciprocal pineal endocrine relations should be repeated, taking into account the latest findings related to the correlations with the diencephalic neurosecretory centers, the hypophysis, the thyroid and parathyroid, the pancreas, the adrenals, and the gonads. The acetylcholinesterase of the diencephalic neurosecretory centers intervenes in some manner in the regulation of the level of antidiuretic hormone, liberated by the neurohypophysis.

Contents

THE PINEAL GLAND

A. Anatomical and Physiological Survey

B. Enzymic Activity

(a). Phosphatases

(b). Carboxylic Esterases

(c). Peptidases

(d). Succinic Dehydrogenase and Succinic Oxidase

(e). Phosphorylase

(f). Other Enzymic Activity

1 β-glucuronidase

2 Monoamine oxidase

3 Decarboxylase

4 Methyltransferases

C. Discussion

(a). Serotonin

(b). 5-Hydroxy and 5-Methoxyindole Acetic Acids

(c). Melatonin

Bibliography

THE NEUROSECRETORY SYSTEMS

IN THE INVERTEBRATES

A. Histophysiological Summary

B. Enzymology

1 In the Molluscs

2 In Insects

(i). In the brain

(ii). In the pars intercerebralis–corpus allatum system

(iii). In the prothoracic gland

IN THE VERTEBRATES

THE HYPOTHALAMO-HYPOPHYSEAL COMPLEX

A. Anatomical and Physiological Survey

(i). The Hypothalamo-neurohypophyseal System

1 The existence of possible neurosecretory centres other than the pre-optic, supra-optic and paraventricular nuclei

2 The unity or plurality of the secretory principles

3 Change in the secretion during its passage

4 Significance of the time of appearance of the neurosecretion in different centres

5 The exclusive production of one hormone by a centre

6 The importance of the number of neurosecretory axons ending in the median eminence

7 The poor understanding of the hypothalamic-anterior pituitary connections

8 The significance of synapses

(α). Excitatory synapses

(β). Inhibitory synapses

(ii). The Adenohypophysis

1 Pars intermedia

2 Pars distalis

(a). Cells elaborating growth of somatotrophic hormone

(b). Gonadotrophic follicle-stimulating cells

(c). Gonad-stimulating luteinising cells

(d). Thyrostimulin or thyrotrophic hormone cells

(e). Prolactin cells

(f). Adrenocorticotrophic cells

3 Pars tuberalis

4 Adenohypophyseal sexual dimorphism

5 Persistent adenohypophyseal enigmas

(α). Cone of Wulzen

(β). The pharyngeal hypophysis or orohypophysis

B. Enzymic Activity

(a). Phosphatases in the Normal Subject

(i). Hypothalamo-neurohypophyseal system

(α). Before birth

(β). After birth

In man

In dog, rabbit and guinea pig

In rat

In birds

Enzymic activity of the synapses

(ii). The adenohypophysis

1 The pars intermedia

2 The pars distalis

(α). In anurans

(β). In mammals

In man

Experimental variations in the phosphatase activity

(i). Hypothalamo-hypophyseal activity

(ii). The adenohypophysis

Discussion

(b). Carboxylic Esterases

(i). Hypothalamo-neurohypophyseal system

Experimental variations

(ii). The adenohypophysis

(α). In poikilotherms

(β). In embryo mammals

(γ). In adult vertebrate

Pars intermedia

Pars distalis

Experimental variations in the carboxylic esterase activity

Discussion

(c). Other Enzymic Activity

1 Peptidase activity

(α). Hypothalamus

(β). Adenohypophysis

2 β-Glucuronidase activity

3 Sulphatase activity

4 Phosphorylase activity

5 Dehydrogenase activity

(α). Hypothalamo-neurohypophyseal system

(β). Adenohypophysis

6 Amine oxidase activities

Conclusion

THE CAUDAL SPINO-HYPOPHYSEAL COMPLEX OF FISH

A. Morphology

B. Histoenzymology

(a). Enzymes Catalysing Oxidations

(b). Phosphatases

(c). Carboxylic Esterases

Bibliography

THE PINEAL GLAND

*

Certainement ce n’est pas un organe glandulaire et on ne doit pas lui accorder une bien grande importance." (Faivre, 1857.)

Sa seule raison d’être est que son ombre projetée sur les radiogrammes peut indiquer le siège d’une tumeur des hémisphères. (Krabbe, 1938.)

… Some function, whatever it may be, must be associated with this complex organ … (Kelly, 1958.)

… The pineal apparatus is an organ concerned with adjustment of internal activities of the animal to correspond to changing conditions of illumination … (Denton, 1965.)

A. Anatomical and Physiological Survey

The gland, which is nearly always present, is an evagination of the roof of the diencephalon†. The form and structure is very variable from one vertebrate to another. The Californian salamander, Taricha torosa, has a filiform gland (400×200 μ) (Kelly, 1958),‡ while that of the tortoise is relatively large, since in a brain 15 mm long it reaches 4 mm in length (Faivre, 1857). The pineal gland in the elephant is the size of a millet seed, while that of Megaptera boops approaches the size of a pigeon’s egg. The pineal of the opossum is extremely small (Jordan, 1911) and the gland is lacking in most edentates (Krabbe, 1938). As early as 1910, Cutore compared the weights of the brain and the pineal gland in a number of mammals. The comparison established clearly the disparity in size. Man, whose brain weighs at least 1300 g, has a pineal gland weighing 220 mg, while the horse, whose brain weighs 512 g, has a pineal gland of 440 mg, and the mule, whose brain is still smaller (430 g), has a pineal gland nearly twice as big (860 mg). The individual variations in the size of the gland are not less than the generic differences and they have been noted by all workers. Recently Giarman and Freedman (1960), in a study on adult humans, reported that the weight of the gland may vary from 0·095 g to 1·890 g. In Bovinae, Ovinae and Suidae there can be a fivefold variation in the size of the gland in animals of the same weight.

Variations of the same order occur in birds. Among the marine species, cormorants (Phalacrocorax pelagicus, P. penicillatus) have the largest pineal glands and petrels (Oceanodroma homochroa) the smallest (Renzoni and Quay, 1963). The stilt bird, Catoptrophorus semipalmatus, of very similar size to P. pelagicus, has a much smaller gland (Quay, 1965). The pineal gland of Strix aluco is rudimentary (Krabbe, 1955) while that of Strix flammea is relatively large (Studnicka, 1905). The emu (Dromiceius novaehollandiae), which is one of the largest birds, has a pineal gland reaching 1 cm in length and weighing 100 mg (Cobb and Edinger, 1962). However, the individual variations are so enormous that the observations could usefully be repeated, not only in birds, but in all vertebrates.

The pineal gland is a solid structure in most vertebrates, but may be hollow as in some teleosts (Argyropelecus olfersi, Maurolicus mulleri). The morphological variations in the teleosts are extreme, however, since the gland is punctiform in Cyclothone, club shaped in Chauliodus and an umbel in Chlorophthalmus, etc. (Holmgren, 1959). The pineal is also hollow in amphibia [(Kamer, 1956–60); Rana esculenta (Kelly, 1960), Hyla regilla (Eakin et al., 1963–4), Xenopus laevis (Van de Kasner, 1962)], in reptiles such as Cordylus polyzonus, Mabuya sulcata (Stein, 1957) and Sceloporus occidentalis (Eakin et al., 1961–2) and in such passeriform birds as Tachycineta thalassina, Sturnella neglecta, Passer domesticus, etc. (Quay and Renzoni, 1964).

The pineal innervation, which has been the subject of longstanding controversies, caused by artefacts obtained with the silver techniques, and numerous speculations on hypothetical epithalamic, epiphyseal and epithalamic-hypophyseal systems, now appears to be established. The pineal gland does not seem to receive fibres from the epithalamus. The glands of large mammals stained with toluidine blue show metachromatic nerve fibres bordering the proximal zone and then turning away sharply (Arvy, 1965). Nevertheless, there is general agreement that the gland is innervated. Cajal (1911) emphasised that, in the rabbit, the pineal nerve plexus was perhaps one of the richest and densest of all glands and that it was formed by fibres derived from the superior cervical sympathetic ganglion. Rat is similar and in this rodent extirpation of the superior cervical ganglion is followed by rapid regression and eventual disappearance of all the pineal nerve fibres (Ariëns Kappers, 1960–5; Pellegrino de Iraldi and Rodriguez de Lores Arnais, 1962). Falck’s admirable technique (1962) enabled Bertler et al. (1963–4) to establish definitely that the pineal innervation is both adrenergic and tryptaminergic. In ultraviolet light, a yellow and a green fluorescence of the pineal cells and nerve fibres can be seen. The former is due to the biogenic amine content and the latter to the catecholamines. All procedures giving a reduction in the concentration of these compounds decreases the fluorescence and conversely procedures leading to an increase in their amount brightens it. Finally, removal of the superior cervical ganglion decreases the normal level of fluorescence, the green disappearing first, followed later by the yellow of the biogenic amines.

A morphology which is so variable from one species to another could reasonably have as a corollary varied functions. The generalisation and extension of observations made on the pineal gland of one vertebrate to those of all species would be hazardous.

Recent histological studies made using the electron microscope in rat (Milofski, 1958; Pellegrino de Iraldi and de Robertis, 1961; Ariëns Kappers, 1961; Gusek and Santaro, 1961; Pellegrino de Iraldi et al., 1964; Clementi et al., 1965), in pig (Heinecke, 1959) and in sheep (Anderson, 1962) do not leave any doubt as to the glandular nature of the pineal.

Morphological changes which arise in the pineal glands of rats treated with reserpine, aldosterone, desoxycorticosterone, cortisone, testosterone, oestrogens and also in castrated animals demonstrate that the pineal gland is capable of responding to these treatments. The work suggests that the gland has some plasticity. The papers reviewed by Clementi et al. (1965) show that after castration the pineal glands of rats become overloaded with ribonucleic acid and also that intense illumination and oestrogen treatment selectively injure the mitochondria, while testosterone causes lipid overloading of the gland. Treatment with aldosterone, desoxycorticosterone or reserpine causes disappearance of the granules in the vesicles of the sympathetic nerve terminals in the pericapillary space shown in Fig. 1. The reactions of the pineal are highly equivocal, being peculiar to the substance used, and they can be demonstrated as various enzymatic changes.

FIG. 1 Nerve terminal in the pericapillary space of the rat pineal gland with two kinds of vesicle: the one is clear, the other granular. ×24,500 (from Clementi et al., 1965)

Apparently the role of the pineal gland, although still much disputed, cannot be essential to life. Numerous vertebrates have been pinealectomised and have survived the procedure. Gladstone and Wakeley (1940) report the case of a soldier in whom a bullet wound had produced a pinealectomy without other injury. Pinealectomy has been carried out in sheep (Dandy, 1915; Dernel, 1927–8; Coglan et al., 1960), dog (Simonnet and Thieblot, 1951; Farrel, 1960), cat (Martin, 1941), rabbit (Exner and Boese, 1910; Sarteschi, 1913–14; Clementi, 1922–3; Yoshizuma, 1931; Imuro, 1937; Baba, 1938; Mezaki, 1939; Nakai, 1950; Nakashita, 1951; Tokumitzu, 1951; Yamasaki and Iwamiya, 1951–5, etc.), guinea pig (Horrax, 1916; Clementi, 1922–3), rat (Izawa, 1926; Anderson and Wolf, 1934; Ikuta, 1938; Martin, 1941; Simonnet and Thieblot, 1951; Kitay, 1954; Holmes, 1956; Wurtzmann et al., 1959; Quay, 1961; Rennels and Dill, 1961), cock (Foa, 1912–29; Izawa, 1922; Shellabarger, 1950–2; Pflugfelder, 1957), two teleosts, Lebistes reticulatus (Pflugfelder, 1953–6) and salmon (Holmgren, 1958), and has been the subject of very contradictory reports in these species. Therefore, if the gland plays an important role other tissues must be capable of compensating for its removal.

Fay’s opinion (1933) that the gland conditions the evolution of all gnoses remains isolated and supremely optimistic. It seems improbable that ingestion, any more than injection, of pineal extract could make humans more intelligent.

A series of experiments involving resorption of the pineal gland, brought about by injection of pineal extracts or grafts of pineal glands, do not offer any explanation as to the function of the gland. The present experimental data are an inextricable mixture of inconsistencies and contradictions and until purified extracts of the active principles are obtained there appears no hope of resolving the problem.

B. Enzymic Activity

The enzymic activity of the pineal gland has been investigated in monkey (Macaca mulatta), horse, Bos taurus L., Ovis aries L., Sus scrofa L., goat, cat, rat and some other rodents (Sciuridae, Heteromyidae, Cricetidae) (Wislocki and Dempsey, 1948; Mikami, 1952; Leduc and Wislocki, 1952; Shimizu and Morikawa, 1957; Shimizu and Okada, 1957; Giarman and Day, 1958; Quay, 1958–60; Niemi, 1960; Arvy, 1961).

All the investigations have established that the pineal gland of young, healthy animals is supplied with a profuse vascular network which hydrolyses adenosine triphosphate and sodium glycerophosphate rapidly, in an alkaline medium. The pineal cells are rich in acid Phosphomonoesterase, Phosphorylase, succinic dehydrogenase, leucyl β-naphthylamidase, β-glucuronidase and in tryptamine oxidase. Some of the pineal vessels are accompanied by fibres which hydrolyse acetylcholine (Fig. 2).

FIG. 2 Appearance of the ram pineal gland after demonstration of glycerophosphatase activity (Arvy, 1961)

(a) PHOSPHATASES

The first investigations, made after fixing with 80% alcohol at pH 9·5 and with long incubation (24 hr), showed that the pineal gland of Macaca mulatta is rich in phosphatases. Fructose diphosphate is hydrolysed rapidly, the adenylic acid of yeast less strongly and sodium glycerophosphate at a still lower rate (Wislocki and Dempsey, 1948). In sections made with a cryostat and fixed in absolute acetone for 30 min, or in paraffin sections of the gland fixed for 24 hr in acetone, the vessels of the pineal gland show significant hydrolysis of sodium glycerophosphate when incubated for 1 hr in an alkaline medium, whereas the nuclei of the gland cells show a similar strong reaction in an acid medium (Leduc and Wislocki, 1952).

In frozen sections of the pineal gland of calf, sheep or pig, fixed in cold 10% neutral formalin for a short time, the alkaline Phosphomonoesterase activity is selectively concentrated in the walls of certain vessels and is very strong since they are stained in 20 min (Fig. 2). The same picture is obtained when adenosine triphosphate (method, Wachstein and Meisel, 1957) is substituted for sodium glycerophosphate (method, Gomori, 1939–55). In both cases small islets of pineal cells are seen enclosed in a fine vascular network charged with phosphatase.

One of the characteristics of the pineal gland is the high concentration of glycerophosphatase in certain vessels. The enzyme has recently been found by Lierse (1965) in Anolis carolinensis.

Some recent biochemical work (Jouan and Rocaboy, 1965) showed clearly that Phenolphthalein phosphate can be hydrolysed by the pineal gland of pig, much more strongly than by the neighbouring cerebral tissue. In homogenates, these workers found that (at pH 9·5) pineal tissue liberates 31 ± 3 μg while the cerebral tissue only liberates 2·1±0·5 μg.

When working at an acid pH, rather than an alkaline one, there are pineal cells which hydrolyse sodium glycerophosphate strongly (method, Gomori). Here the pineal gland appears dark in the middle of the roof of the diencephalon which is much less rich in histochemically detectable Phosphomonoesterase.

(b) CARBOXYLIC ESTERASES

Since 1952 Leduc and Wislocki have observed that the pineal gland can hydrolyse Tween 40 and 60 and also β-naphthyl acetate. However, the pineal carboxylic esterase activity, studied on frozen sections, seems weak, particularly when compared with the activity of the surrounding diencephalon. When α-naphthyl acetate (method, Burstone), butyryl or acetylthiocholine (method, Koelle) are used the pineal gland always appears lighter than that part of the diencephalon which lies close to it.

The use of acetylthiocholine in calf, sheep and pig reveals fibres embedded in the vessel walls (Fig. 3). The distribution of the vessels having such fibres shows that the pineal glands of these three artiodactyles have a double symmetry, bilateral and dorsoventral. The vessels of the posteroventral zone are more deeply stained than those of the anterodorsal zone.

FIG. 3 Appearance of the pineal gland of ram after demonstration of acetylthiocholinesterase activity (Arvy, 1961)

(c) PEPTIDASES

The pineal gland is one of the endocrine organs richest in peptidases, at least in the rat (Niemi, 1960) and in some large domestic animals, bovines, ovines and porcines (Arvy, 1961). Under the experimental time conditions which are such that the rest of the brain of rat (Niemi) and the roof of the diencephalon of large mammals (Arvy) do not show peptidase activity, the pineal gland rapidly breaks down leucyl β-naphthylamide (method, Nachlas et al., 1957).

Using o-acetyl-5-dinitrophenol phosphate (= E 600) as inhibitor, Niemi’s (1960) very precise research has established that the peptidase activity of the pineal gland is of the cathepsin C type.

(d) SUCCINIC DEHYDROGENASE AND SUCCINIC OXIDASE

Leduc and Wislocki (1952), Shimizu et al. (1957a, b), Quay (1958–60) and Arvy (1961) have investigated the succinic dehydrogenase activity of the pineal gland (method, Seligman and Rutenburg, 1951; Rosa and Velardo, 1954; Farber and Louviere, 1956; Nachlas et al., 1957) in some rodents (rabbit, guinea pig, mouse and desert rodents) and in three artiodactyles.

Quay (1958) has made a comparative cytochemical and biochemical study on a thousand rats. He noted that the pineal succinic oxidase activity increased rapidly during the first 6 weeks of life to attain an activity more than twice as great as that at birth, from 60 to 150 (μmoles tetrazolium reduced per μg dry weight gland). In the adult rat, this activity is very stable; castration, injection of sex hormones, parathormone, thiourea, thyroxine, corticosteroids and many other substances do not affect it.

In sections of rat brain, or in sections of the roof of the diencephalon of Bos taurus L., Ovis aries L. and Sus scrofa L., the classical stains for succinic dehydrogenase are taken up preferentially by the gland.

Quay (1960) compared the succinic dehydrogenase activity of the ependymal choroidal complex and the pineal gland of various Sciuridae, Heteromyidae and Cricetidae, as well as in Oryctolagus cuniculus and Felis catus. The activity of the pineal gland is always less than that of the ependymal choroidal complex. The difference may be very small; for example, in Eutamias speciosus (Sciuridae) it is 100 to 108, but in Dipodomys elephantinus (Heteromyidae) the activity reaches 159 (μmoles triphenyltetrazolium chloride reduced per μg dry weight gland) in the complex while it is not more than 88 in the pineal gland. These adjacent structures do not therefore have the same succinic dehydrogenase activity. Moreover, the evolution of the enzymic activity in the two structures is different. The succinic dehydrogenase activity of the ependymal cells increases rapidly after birth reaching the level of activity characteristic of the adult in two weeks, while the activity in the pineal gland increases only slowly after birth.

In the adult rodent there is no apparent relationship between the succinic dehydrogenase activity of the pineal gland and the resistance of the animals to dehydration or their ability to live in the desert (Quay, 1960). There is also no correlation between the enzyme activity and the ascorbic acid concentration in the gland, at least in rat. Using the Bacchus reaction (Prop and Kappers, 1961) the enzyme can be estimated from the few granules seen distributed unevenly within the pineal cells. In fact the succinic dehydrogenase activity is distributed unevenly through the whole gland.

(e) PHOSPHORYLASE

Shimizu and Okada (1957) obtained a moderate or strong Phosphorylase reaction (method, Takeuchi et al., 1955) in the pineal gland and in the sub-commisural organ of the new-born or 24-hr-old rat. These authors have observed that the Phosphorylase activity in the pineal gland of rat decreases from the fourteenth day of life, so that in the adult there is no histochemically detectable enzyme activity.

The situation is entirely different in the artiodactyles (Arvy, 1961). The pineal gland in bovines, ovines and porcines has a strong Phosphorylase activity. The gland can be distinguished immediately since it is brown in the middle of the straw-coloured roof of the diencephalon.

(f) OTHER ENZYMIC ACTIVITY

1. β-GLUCURONIDASE

The pineal glands of three artiodactyles belonging to the genera Bos, Ovis and Sus have a β-glucuronidase activity which is histochemically detectable (Arvy, 1963).

2. MONOAMINE OXIDASE

The pineal glands of Bos, Ovis and Sus oxidise tryptamine but are inactive on tyramine and histamine (Arvy, 1961).

Tryptamine oxidase activity has been found in the rat (Smith, 1963; Hakanson and Owman, 1965). Three weeks after cervical sympathectomy it is just as strong (Hakanson and Owman, 1965) and fairly intense illumination has no effect (Axelrod et al., 1964). The same enzymic activity is found in rat pinealocytes cultured in vitro (O’Steen and Dill, 1964).

Wurtman et al. (1964, p. 299) found a monoamine oxidase activity in man which was more or less constant between the ages of 3 and 70. In spite of fibrosis and calcification appearing during aging, the pineal gland remains able to oxidise biogenic amines.

A powerful monoamine oxidase inhibitor, the (benzyl carbamoyl ethyl) hydrazide of isonicotinic acid, increases the serotonin content of the gland (Owman, 1964; Jouan, 1965).

3. DECARBOXYLASE

Apparently the pineal gland contains neither histaminase nor histidine decarboxylase (Giarman and Day, 1958). Biochemical techniques have been used to demonstrate a 5-hydroxytryptophane decarboxylase in the pineal glands of bovines (Giarman and Day, 1958).

The pineal gland of rat is remarkably rich in DOPA decarboxylase. This activity is as strong in the pineal as in the kidney (Håkanson and Owman, 1965). The 5-hydroxytryptophane decarboxylase activity fluctuates diurnally and with the oestrus cycle in rat when the superior cervical ganglion, the eye and the pituitary gland have been removed. This activity plays an essential role in the metabolism of the pineal gland since it controls, for a large part, the presence of serotonin, the precursor of melatonin. It has not yet been the subject of histochemical research.

4. METHYLTRANSFERASES

We know nothing of the histochemistry of these important transferases. They bring about the fixation and removal of methyl groups and thus control the activity of the biogenic amines. Hydroxy-O-methyltransferase is localised exclusively in the pineal gland in hen, cat, cow and macaque monkey. This transferase differs from others known at present. The enzyme is only active in the presence of adenosyl methionin and seems to play a key role in the biosynthesis and inactivation of biogenic amines and their derivatives. In fact, melatonin can be formed simply by putting together an extract of cow pineal gland, N-acetyl serotonin and S-adenosyl methionin (at pH 8 for 2 hr at 37° C). The pineal gland therefore contains an enzyme system capable of transferring the methyl group of S-adenosyl methionin to the hydroxyl group of N-acetyl serotonin. The pineal gland of monkey has the same capacity (Axelrod and Weissbach, 1960).

C. Discussion

The list of enzymic activity in the pineal gland, though barely outlined, may suggest a number of functions.

1. It is likely that the strong Phosphorylase activity of the pineal gland controls its extraordinarily rapid phosphate turnover, which was discovered by Borel and Orström (1945–7) and confirmed by Reissel et al. (1949) and Brewer and Quay (1958) in the rat. The use of radioactive phosphorus shows that the pineal gland plays an important role in cerebral phosphate metabolism. The specific activity in the pineal is 26·5 compared with 0·69 in the corpora quadrigemina, 0·78 in the thalamus and 2·68 in the habenula (Borell and Orström, 1947). In the female rat, the individual variations in the specific activity can be very large (28–66) but the pineal activity is always considerably stronger than that of the hypophysis: values of 46·33 in the pineal compared with 5·21 in the pars distalis and 10·89 in the pars nervosa have been quoted (Brewer and Quay, 1958).

In this connection, knowledge of the evolution of pineal phosphatase activities after hypophysectomy would be important, since the ability to store radioactive phosphorus and iodide is greatly increased by this procedure. Reiss et al. (1949) have shown that the pineals of rats administered 2 μc of ³¹P and ¹³¹I give respectively 381–333–280 and 634–704–820 c.p.m. in normal rats and 1028–728–644 and 1420–1285–1122 in hypophysectomised rats.

2. Some anomalies in phosphate turnover might reasonably be related to calculus degeneration which perhaps has its seat in the pineal gland. The calculi are always present in old bovines and are sometimes very numerous (Fig. 3A). They were noted by the first anatomists who gave them the name acervulus cerebri or cerebral sand (Faivre, 1857; Clarke, 1860). They have been reported in man and I have found them in the pineal gland of multiparous ewes. Old age is not the determining factor in their appearance; in fact, pineal gland removed from young calves (less than 3 months old), reared on such an unbalanced diet that their growth was severely handicapped, all contain numerous calculi. Stammer (1961) has described such calculi in the pineal glands of Ardea cinerea and Anser albifrons. Tiberin and Beller (1963), Quay (1965) and Heiniger (1965) have recently studied them in the pineal glands of humans.

3. Pineal carboxylic esterases must be implicated in lipid metabolism. This metabolism, which is quite important, can be demonstrated by biochemical techniques but is not obvious in histochemical studies. Nevertheless, in frozen sections, oil red shows minute lipid inclusions in the pineal cells of man, bovines, hamster and rat (Quay, 1961). In relation to this, continuous illumination (for at least 4 weeks) depletes the pineal cells of their lipids (from 19 to 14% in rat and 28 to 24% in hamster), while the succinic dehydrogenase activity decreases (Quay and Halevy, 1962). On the other hand, a period in the dark causes an increase in the histochemically detectable lipids in the pineal gland of Rattus norvegicus and Mesocricetus auratus (Quay, 1961). It would certainly be interesting to know the behaviour of carboxylic esterases in the pineal glands of animals reared under different conditions of illumination, especially since Farrell (1960) was able to isolate from the pineal gland of the dog an active lipid-soluble principle, adrenoglomerulotrophin, which could selectively increase the secretion of aldosterone, without affecting that of cortisone. However, pinealectomy in the rat does not interfere with the secretion of aldosterone (Wurtman et al., 1960) and it does not cause a modification of the sudanophilic lipids, cholesterol or the adrenal esters of cholesterol (Rennels and Dill, 1961).

Treatment with desoxycorticosterone increases pineal lipids (Hungerford and Panagiotis, 1962). After ovariectomy also the lipid content of the pineal gland of rat increases (Zweens, 1963) but adrenalectomy decreases these lipids (Hungerford and Panagiotis, 1961).

That the histochemically detectable lipids in the pineal gland are osmophilic and sudanophilic has been known since the beginning of the century. They also stain with acid haematin (Quay, 1957).

Recent research has shown clearly the triglyceridic nature of some pineal lipids. The triglycerides are associated with choline and phospholipids (Prop, 1963). The latter constitute 32% of the total lipids and, of this fraction, about 50% are phosphatidylcholins, 27% cephalins of ethanolamine and serine, 12% acid phosphatides and 11% sphingomyelins (Jouan et al., 1964, p. 1121; Tu Viêm Dai, 1964). In pig, the total pineal lipids contain 16% cholesterol (Tu Viêm Dai, 1964).

4. The presence of an active peptidase in the pineal gland suggests that peptides are elaborated inside the pineal cells.

5. Without doubt the Phosphorylase activity is connected more with the metabolism of the pineal cells themselves than with the synthesis of glycogen, for the Phosphorylase activity is strong when the glycogen content is relatively poor. The Bauer–Feulgen reaction does not show trace of glycogen, but the MacManus reaction does, at least in the rat pineal gland. This reaction makes some PAS + granules visible, which are removed from the sections by saliva (Leduc and Wislocki, 1952). However, in the rhesus monkey (Wislocki and Dempsey, 1948), and in horse, goat and pig (Mikami, 1951), two kinds of cells can be distinguished in the pineal gland. One has no histochemically detectable glycogen while the other contains this glucide. It may also be noted that the pineal gland is remarkably rich in neuraminic acid (Green et al., 1962).

In general, the postnatal development of the cerebral phosphatase in the rat is such that some intimate role for the enzyme in the metabolism and function of the brain can rationally be envisaged (Shimizu and Okada, 1957).

6. The most recent research gives rise to ideas of a different order which could form the basis for an advance in pineal histochemistry.

(a) SEROTONIN

Giarman et al. (1958–60) and Miline et al. (1959) have established that the pineal gland of rhesus monkey, cow and hare are extremely rich in histamine, catecholamines, acetylcholine and serotonin. The pineal gland of bull contains 10 μg/g histamine, but histaminase or histidine decarboxylase could not be detected. On the other hand, the metabolism of 5-hydroxytryptamine seems to be important in the pineal for the enzyme involved in the synthesis of serotonin, hydroxytryptophane decarboxylase, is present together with an enzyme for its breakdown, monoamine oxidase (Giarman and Day).

Assayed on Venus mercenaria, serotonin is apparently much more concentrated in the pineal gland of man and monkey than that of cow. The individual variations are, however, very great. The pineal gland of cow contains on average 0·40 μg/g (0·20–0·63 μg/g) while that of the adult male rhesus monkey contains 3·28 μg/g and the serotonin content of the pineal in man varies between 0·36 and 22·82 μg/g. There is no correlation between the gland weight and the serotonin content in man; a pineal gland which weighs 1·89 g can contain 0·36 μg/g serotonin while a gland weighing 0·095 g can contain 2·63 μg/g (Giarman et al., 1960). The serotonin content of the pineal gland is always much greater than that of the brain; in the hare, when the pineal contains 0·26 μg/g, the rest of the brain has 0·075 μg/g (Miline et al., 1959).

Serotonin seems to be present in the pineal gland of all vertebrates. The pineals of the chelonians, Terrapena Carolina and Gopherus berlandieri, contain respectively 6·98 and 9·84 μg/g serotonin. The pineal of the lacertilian, Sauromalus varius, seems relatively poor in serotonin (0·87 μg/g), but that of Gerrhonotus multicarrinatus has a high concentration (41 μg/g). The ophidians, Natrix sipedon and Pituophis catenifer, contain respectively 10 and 3·8 μg/g serotonin (Quay and Wilhoft, 1964).

The pineal serotonin concentration is subject to diurnal variations. In cats of either sex kept under normal conditions of illumination, the serotonin content decreases from 6 p.m. to 10 p.m. and increases again from 10 p.m. to 12 a.m. Moreover, the pineal serotonin fluctuates during the course of the oestrus cycle, varying from 15 to 17 nanograms (Quay, 1954, p. 61).

The electron microscope shows that the pineal biogenic amines can be localised. In fact the pineal cells of rat, magnified 48,000 times, show multivesicular bodies, which disappear after the administration of a serotonin liberator such as reserpine (Pellegrino de Iraldi and De Robertis, 1961). The inclusions can be modified in several ways (Clementi et al., 1965). An important part of the serotonin is localised in the sympathetic nerve terminal (Fig. 3) (Quay and Halevy, 1962; Bertler et al., 1963), for removal of the superior cervical sympathetic ganglion is followed by loss of the pineal nerve plexus, while the serotonin content of the gland decreases (Bertler et al., 1963; Pellegrino de Iraldi et al., 1963).

(b) 5-HYDROXY AND 5-METHOXYINDOLE ACETIC ACIDS

The pineal gland of bovines contains other compounds related to serotonin. Working on 200,000 bull glands, Lerner et al. (1958–61) were able to isolate three, very closely related, substances which all gave the same blue colour, characteristic of indole compounds, when they were added to p-dimethylaminobenzaldehyde (Ehrlich’s test, 1901). Two of them are present in equal concentrations (200 μg/g, fresh tissue). They can be identified as 5-methoxyindole-3-acetic acid and 5-hydroxyindole-3-acetic acid. They do not have any action on melanocytes. The third substance, however, is much less plentiful (40 μg 100 g) but is extremely active on melanocytes.

Diurnal variations do not occur in the 5-methoxyindole acetic acid content of the pineal gland, which contains about 0·098 μg. On the other hand, 5-hydroxyindole acetic acid is subject to daily and oestral variations similar to those of the serotonin content. In the adult female rat the variation is 4 μg at night to 18 μg in the afternoon, decreasing when the rats are submitted to darkness (Quay, 1964, p. 710).

(c) MELATONIN

This substance is absolutely specific to the pineal gland: the hypophysis, hypothalamus, thymus, adrenal, ovary, testis, eye, etc., contain nothing equivalent to melatonin (Lerner et al., 1958). This substance does not act on the uterus, like adrenaline or noradrenaline, nor affect Venus mercenaria heart, like serotonin. The cutaneous melanocytes of Rana pipiens are extremely sensitive to Lerner’s melatonin since 10−12 g are sufficient to prevent or reverse the effect of intermedin, adrenocorticotrophic hormone or caffeine on melanocytes.

Lerner’s melatonin is 100 times more active than adrenaline, 200 times more active than tri-iodothyronine and 5000 times more active than serotonin in preventing the action of intermedin on the cutaneous melanocytes of Rana pipiens (Lerner et al., 1958).

Since it is too scarce to be extracted from the pineal gland in useful quantities for experimentation, melatonin has been obtained by synthesis from 5-methoxyindole-3-acetonitrile. The compound obtained by synthesis and the pineal compound have the same chemical and physical properties (the same fluorescence curves, UV absorption spectra and behaviour during electrophoresis and chromatography). As a result it has been established that the pineal glands of bovines contain N-acetyl-5-methoxytryptamine; this is the active principle, capable of blanching the skin of anurans and fishes, which has intrigued physiologists since the observations of von Frisch (1910).

The three pineal indole derivatives, identified by Lerner et al. (1958–61), seem to be derived from serotonin, by acetylation and methylation, followed by hydroxylation and oxidation (Lerner et al., 1960).

Assayed on Xenopus larvae the pineal gland of rat contains 5 × 10−4 μg melatonin, or 0·4 μg/g (Prop and Kappers, 1961), about twice as much as that of bull. However, extracts of bull pineal glands on chromatograms give a beautiful blue spot with Ehrlich’s reagent and this test, in vitro, can detect the presence of 0·04 μg melatonin. Nevertheless, all the histochemical tests for indole compounds (Lillie, Glenner-Lillie, Fischer-Lillie, Ehrlich) made on the rat pineal gland are negative (Prop and Karpers, 1961). However, the pineal glands of sow and ram are considerably richer in aromatic amino acids than the rest of the roof of the diencephalon which border them. Indeed, when Glenner’s reaction using rosindole is applied, they appear dark blue, the roof of the diencephalon being blue-grey (Arvy, 1963).

The detection of N-acetyl-5-methoxytryptamine, 5-hydroxyindole-3-acetic and 5-methoxyindole-3-acetic acid in situ would obviously be important. The definition of the factors determining their concentration and the better understanding of the function of the pineal gland would be possible.

Microelectrophoresis in polyacrylamine gel (Pun and Lombroso, 1964) enables seventeen fractions to be separated from the rat pineal gland. This method seems particularly suitable to show the experimental variations in the components of the gland in rat.

In conclusion. The work of the last 5 years has made more contributions towards the understanding of the pineal gland than that of the two preceding centuries. The study of all the reciprocal pineal endocrine relations should be repeated, taking into account the latest findings related to the correlations with the diencephalic neurosecretory centres, the hypophysis, the thyroid and parathyroid, the pancreas, the adrenals and the gonads.

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2. HlSTOPHYSIOLOGICAL SUMMARY

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