Table Of ContentHISTOENZYMOLOGY
OF THE ENDOCRINE GLANDS
BY
L. ARVY, D.M., D.Sci.
Histoenzymological Laboratory, Faculty of Medicine, Paris VI
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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 histoche-
mically 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 charac-
terising the endocrine role of certain structures lacking ducts, such as the spleen, thymus,
lymphatic nodes, Fabricius pouch, etc. Further biochemists have not succeeded in iso-
lating a specific active principle. Therefore, I am only dealing with histochemically
detectable enzymic activity in glands associated with the vascular system whose en-
docrine 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 argu-
ing 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.
vii
INTRODUCTION
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 Mile 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. Mile Arvy, in addition to considerable activity in
original scientific research, attacked this task with rare good humour. Several years ago
Mile 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,
Mile 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.
LUCIEN LISON
ix
CHAPTER I
DIENCEPHALIC ENDOCRINE GLANDS
Contents
THE PINEAL GLAND 3
A. Anatomical and Physiological Survey 3
B. Enzymic Activity 6
(a) Phosphatases 7
(b) Carboxylic Esterases 8
(c) Peptidases 9
(d) Succinic Dehydrogenase and Succinic Oxidase 9
(e) Phosphorylase 10
(f) Other Enzymic Activity 10
1. ^-glucuronidase 10
2. Monoamine oxidase 10
3. Decarboxylase 11
4. Methyltransferases 11
C. Discussion 11
(a) Serotonin 13
(b) 5-Hydroxy and 5-Methoxyindole Acetic Acids 14
(c) Melatonin 15
Bibliography 16
THE NEUROSECRETORY SYSTEMS 33
IN THE INVERTEBRATES 33
A. Histophysiological Summary 33
B. Enzymology 37
1. In the Molluscs 37
2. In Insects 37
(i) In the brain 37
(ii) In the pars intercerebralis-corpus allatum system 37
(iii) In the prothoracic gland 38
IN THE VERTEBRATES 38
THE HYPOTHALAMO-HYPOPHYSEAL COMPLEX 38
A. Anatomical and Physiological Survey 38
ι
2 HISTOENZYMOLOGY OF THE ENDOCRINE GLANDS
(i) The Hypothalamo-neurohypophyseal System 3 S
1. The existence of possible neurosecretory centres other than the pre-optic, supra-optic and
paraventricular nuclei 43-
2. The unity or plurality of the secretory principles 43
3. Change in the secretion during its passage 44
4. Significance of the time of appearance of the neurosecretion in different centres 44
5. The exclusive production of one hormone by a centre 44
6. The importance of the number of neurosecretory axons ending in the median eminence 45
7. The poor understanding of the hypothalamic-anterior pituitary connections 46
8. The significance of synapses 47
(a) Excitatory synapses 45
(β) Inhibitory synapses 52
(ii) The Adenohypophysis 52
1. Pars intermedia 53
2. Pars distalis 55
(a) Cells elaborating growth of somatotrophic hormone 59-
(b) Gonadotrophic follicle-stimulating cells 61
(c) Gonad-stimulating luteinising cells 62
(d) Thyrostimulin or thyrotrophic hormone cells 65
(e) Prolactin cells 66
(f ) Adrenocorticotrophic cells 69·
3. Pars t überall s ΊΖ
4. Adenohypophyseal sexual dimorphism 73
5. Persistent adenohypophyseal enigmas 73
(a) Cone of Wulzen 74
(β) The pharyngeal hypophysis or orohypophysis 74
B. Enzymic Activity 75
(a) Phosphatases in the Normal Subject 76
(i) Hypothalamo-neurohypophyseal system 76
(a) Before birth 76
(β) After birth 77
In man 77
In dog, rabbit and guinea pig 78
In rat 78
In birds 80
Enzymic activity of the synapses 80
(ii) The adenohypophysis 81
1. The pars intermedia 81
2. The pars distalis 82
(a) In anurans 82
(ß) In mammals 82
In man 82
Experimental variations in the phosphatase activity 83
(i) Hypothalamo-hypophyseal activity 83
(ii) The adenohypophysis 84
Discussion 87
(b) Carboxylic Esterases 85
(i) Hypothalamo-neurohypophyseal system 88-
Experimental variations 90
(ii) The adenohypophysis 91
(a) In poikilotherms 91
(ß) In embryo mammals 91
(γ) In adult vertebrate 92
THE PINEAL GLAND 3
Pars intermedia 92
Pars distalis 94
Experimental variations in the carboxylic esterase activity 94
Discussion 95
(c) Other Enzymic Activity 96
1. Peptidase activity 96
(a) Hypothalamus 96
(β) Adenohypophysis 96
2. ^-Glucuronidase activity 97
3. Sulphatase activity 98
4. Phosphorylase activity 98
5. Dehydrogenase activity 98
(a) Hypothalamo-neurohypophyseal system 98
(β) Adenohypophysis 99
6. Amine oxidase activities 100
Conclusion 101
THE CAUDAL SPINO-HYPOPHYSEAL COMPLEX OF FISH 102
A. Morphology 102
B. Histoenzymology 105
(a) Enzymes Catalysing Oxidations 105
(b) Phosphatases 106
(c) Carboxylic Esterases 107
Bibliography 107
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 dien-
cephalon*. The form and structure is very variable from one vertebrate to another.
The Californian salamander, Taricha torosa, has a filiform gland (400x 200 μ) (Kelly,
1958),î while that of the tortoise is relatively large, since in a brain 15 mm long it reaches
* = épiphyse = conarium = glandula pinealis = glandula turbinata = turbo = "pine nut" =
"Man's yard" = Zirbeldrüse, etc.
t In the humming birds, Cholorostilbon and Eupetoma, the pineal gland is not connected to the di-
encephalon (Krabbe, 1955). In the hooded rat the pineal is separated from the diencephalon by a sup-
porting band of connective tissue. Rostrally the gland is connected to the choroid plexus and caudally
to the inferior surface of the transverse sinus (Gardner, 1958). Sirenians do not have a pineal gland
(Dexler, 1913).
J Each observation reported is attributed to its author; the bibliography mentions only the literature
dealing directly with the subject.
4 HISTOENZYMOLOGY OF THE ENDOCRINE GLANDS
4 mm in length (Faivre, 1857). The pineal gland in the elephant is the size of a millet
seed, while that of Megaptera hoops 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 eden-
tates (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, Ca~
toptrophorus 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 novae-
hollandiae), 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 mulled). The morphological variations
in the teleosts are extreme, however, since the gland is punctiform in Cyclothone, club
shaped in Chauliodus and an umbel in Chlor ophthalmus, 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 neg-
lecta, 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 ap-
pears to be established. The pineal gland does not seem to receive fibres from the epi-
thalamus. The glands of large mammals stained with toluidine blue show metachro-
matic 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 su-
THE PINEAL GLAND 5
perior cervical ganglion is followed by rapid regression and eventual disappearance of
all the pineal nerve fibres (Ariens 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 tryp-
taminergic. 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; Ariens 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 re-
serpine, 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 overload-
ing 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.
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; dementi,; 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 ; Rerinels and Dill, 1961), cock
6 HISTOENZYMOLOGY OF THE ENDOCRINE GLANDS
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. X24,500 (from Clementi et al, 1965)
<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 improbabl e 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
mulatto), 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 ani-
mals is supplied with a profuse vascular network which hydrolyses adenosine triphos-
phate and sodium glycerophosphate rapidly, in an alkaline medium. The pineal cells
