ABSTRACT
The object of this investigation is to throw further light upon the relationship of the cortex suprarenalis or inter-renal tissue with the interstitial cells of the testes.
PREVIOUS INVESTIGATION
A. Cortex Suprarenalis
It is generally recognised that both structures are developed from the epithelium of the genital ridge and a mass of evidence has now accumulated to show that the cells of the cortex and the interstitial cells exercise a similar influence on development and growth.
Glynn (11) in a very full account of the relationship of changes in the suprarenal to abnormal conditions of the gonads, states that adrenal hypernephroma in children is invariably characterised by precocious growth of the body generally and of the sexual organs in particular. Stilling (25) found enlargement of the adrenals in male rabbits during the breeding season. He also observed seasonal variations in the adrenals of frogs during the summer; the peripheral part of the cortex contained peculiar elements—summer cells—which atrophied when the sex glands began to enlarge. Aichel(1) noted that the adrenals were very large in animals with well-developed sex organs or reproductive instincts. He also found that there was an increase in the size of the adrenals of birds and some amphibia during the breeding season. Guieysseta(13) found considerable enlargement of the adrenals affecting especially the zona fasciculata in pregnant guinea-pigs. Gottschau(12), working on pregnant rabbits, stated that the outer part of the cortex increased in thickness. Schenker(24) found enlargement after castration especially in the zona Tasciculata and Cecea (7) stated that he found that the cortex and medulla enlarged after castration. Tamura (30) found in pregnant mice that the gland becomes reduced in area and that there is hypertrophy of the glomerulosa and fasciculata and that the reticularis degenerates.
Elliot and Armour (9) state that the large size of the human foetal suprarenal gland is due to a peculiar hypertrophy of the inner zone of the cortex which commences very early and continues until birth; this inner hypertrophied mass is richly supplied with blood-vessels, but does not contain the fatty substance which is a characteristic product of the adult cortex. They say that this foetal cortex degenerates immediately after birth, undergoing a fatty change, and that at the end of the first year all trace of it has disappeared and that then a layer of small cells, which early in foetal life enveloped this mass, assumes the appearance of the adult cortical cells and stores up fatty substance, develops steadily and, alone, forms the adult cortex.
The structure of the lipoid containing cells of the inter-renal tissue is indistinguishable by staining methods from that of the interstitial cells of the testes and ovaries.
In a previous investigation I have shown that in the mole (28), while no marked change in the size of the suprarenals is manifest, changes in the amount of lipoids in the cortical cells occur throughout the oestrous cycle. As these variations coincided with changes in the testes, this observation suggested that the inter-renal cells play an ancillary part to the interstitial cells of the testes in the storage of lipoids which seem to be required in the production of spermatozoa.
An inter-relationship of the suprarenals and thymus has been demonstrated by Jaffe (16) and between suprarenals, gonad, thymus and thyroids by Marine (17).
Marine has suggested that the inter-renal may be called the para-sex tissue.
B. Testes
Bouin and Ancel (5) have described changes in the interstitial cells of the testes of the horse resembling those found by Armour and Elliot in the cortex suprarenalis of the human subject.
They state that in the foetal horse the interstitial cells are abundant, the tubules being well separated by them. Those cells which contain fatty substances they called xanthochrome interstitial cells. At puberty, the spermatogenic stage, the young interstitial gland disappears and, what the authors call, the true gland forms.
The tubules become arranged into zones, spermatogenesis commencing in the centre. The interstitial cells disappear slowly during the establishment of spermatogenesis. The xanthrochrome cells disappear first in the central region of the testicle, persisting longer at the periphery. In the adult animals the interstitial gland forms large vascular columns, the cells containing pigment granules. In the old animals spermatogenesis is slowed down, and a certain number of tubules are in process of atrophy and disappearance. The interstitial gland has diminished in volume from atrophy of the interstitial cells.
The relationship of the interstitial cells of the testes to the development of the secondary sexual characters is definitely established. Their relationship to spermatogenesis has been investigated in different species by different observers with somewhat contradictory results.
Hansemann (14) in the marmot, Rasmussen (21) in the woodchuck (ground hog), Friedman(10) and Mazzette(20) in the frog and Marshall (18) in the hedgehog found an increase in the number of the interstitial cells during active spermatogenesis. On the other hand, Regaud (23), Tandler and Grosz(26) and le Caillon(6) in the mole, and Champy(8) in the frog found little or no increase of the interstitial cells during the period of active spermatogenesis but an increase as spermatogenesis declined. Reeves (22) described the interstitial cells in the chick as being more numerous at three to five and a half months than later, and Boring and Pearl (4) found the interstitial cells only in the newly hatched chick. I found in the bird (29) that the interstitial cells were most manifest in the pre-oestrous period, and that as spermatogenesis advanced and the tubules enlarged they became less prominent.
Berberich and Jaffe (2) who have examined histologically one hundred human testes discuss the reciprocal relationship between seminal canal and interstitial cells. They regard the lipoids in the cells of Sertoli which may also be laid down in and between the spermatogenic cells and may extend even into the lumen of the canal as physiological and they have determined a definite reciprocal relationship between the amount of this fat and that of the fat in the interstitial cells.
PRESENT INVESTIGATION
In the present investigation the concomitant changes in the testes and suprarenals during development before and after puberty have been studied.
The rat (Mus norvegicus albinus) was chosen because it develops rapidly to sexual maturity, i.e. in about three months. Moreover, the male of this animal does not seem to have a sexual cycle, but breeds at any season and continues sexually active for most of its life if the environment is favourable.
Observations on structural changes and on the lipoid content of the suprarenals and testes were carried out. At least six animals were examined at each of the following ages, (i) two weeks’ embryo, (ii) birth, (iii) 2 weeks, (iv) 4 weeks, (v) 12 weeks, (vi) 18 weeks, (vii) 26 weeks, (viii) 39 weeks, and (ix) sterile old age. The methods of examination were the same as those employed in the investigation on the mole (loc. cit.).
A. Cortex Suprarenalis
(a) Structure
In the embryo the cells of the glomerulosa are small and packed closely together. In the fasciculata and reticularis the cells have large rounded nuclei and the protoplasm is plentiful and vacuolated.
At birth the glomerulosa is little changed in appearance from the embryonic type; the cells forming the fasciculata are closer together, giving a suggestion of the column formation ; few of the cells show vacuoles in the protoplasm. In the reticularis the cells are much more vacuolated than those of the fasciculata and between the cords of cells are wide sinuses.
At 2 weeks of age the cortex of the suprarenals has the normal appearance of an adult cortex. The cells in the deeper layer of the glomerulosa contain distinct round nuclei and are smaller than the other cells of this zone.
By the 4th week the majority of the cortical cells are vacuolated, those of the glomerulosa markedly so.
By the 12th week, the onset of sexual maturity, the cells which formed the ill-defined layer observed in animals of 2 weeks of age, between the glomerulosa and fasciculata are now quite distinctly shown as a definite layer (Plate XIV, fig. 1) ; the cells are smaller and more rounded than even those of the fasciculata and the protoplasm is granular, while that of the adjacent cells is vacuolated. This layer is very distinct and as far as I can find has not been previously described, except possibly by Jackson (15) who only refers to it as a lipoid free zone or transition band. Suprarenals from the dog and rabbit also show this layer; it has in all likelihood been looked upon merely as the deeper layer of the glomerulosa. It will be again referred to when discussing the lipoid content of the suprarenal cortex.
(b) Lipoids
An examination of the sections prepared to demonstrate the presence of lipoids, show that they are evenly distributed over the cortex in the embryonic gland but that by 2 weeks of age the glomerulosa rich in lipoids is separated from the fasciculata by the layer of small cells previously referred to (Plate XIV, fig. 2). These cells are mostly lipoid free. By the 4th week the lipoids are as abundant in the glomerulosa as in the outer part of the fasciculata. Lipoids are also present in the inner part of the fasciculata and reticularis. The band between the glomerulosa and fasciculata is now practically clear of lipoids. During the 12th week the glomerulosa shows a richer staining with sudan than other parts of the cortex and this is the picture given throughout the rest of life. It may be noted that the rat is exceptional in this, as in most animals it is found that the lipoids in the suprarenals are more abundant in the fasciculata than in other layers. There is no suggestion of a definite variation of lipoid in individual sections. Measurements of the organs were not carried out and hence an actual decrease or increase in the total lipoids of the organ could not be determined.
B. Testes
(a) Structure
The testes of the rats were examined at the same ages as the suprarenals. It is found that in the two weeks’ embryo (Plate XIV, fig. 3) the seminal tubules are more distinct at the periphery and that they contain large rounded cells, and between the tubules there is a large amount of fibrous tissue and nests or columns of interstitial cells which cannot be confused with the cells of the fibrous tissue which are present. At birth the tubules are more prominent and the spermatic cells are arranging, themselves around the tube against the fibrous coat ; the spermatogonia are quite distinct; the cells in the centre of the tubules have not got the well-marked nuclei, they form an amorphous mass. There is still a large amount of fibrous tissue and interstitial cells between the tubules which are separated from one another. By the 4th week the tubules are well formed and packed closely together and although no spermatozoa are present there is active spermatogenesis ; the interstitial cells are now fewer in number and lie in some of the angles formed between the tubules ; in some of the tubules there are two rows of regularly packed Small cells with circular nuclei and in the lumen there are large angular cells with very large centrally placed nuclei. On examining sections from animals of 12 weeks of age the tubules give the picture of full spermatogenesis; the spermatozoa are present; the interstitial cells are still seen in some of the angles formed by the tubules. There is no change to be noted from now until sterile old age (Plate XIV, fig. 4) when the tubules again show a separation by wide spaces but no increase in the number of interstitial cells. These wide spaces, Wagner (27) has suggested, are lymph spaces. They appear in the sections of this series at the 4th week and in sections from rats aged 12 weeks (Plate XIV, fig. 5) contain a fluid and certainly have the appearance as noted by Wagner. But on examining the spaces in the testicles of sterile old rats they rather suggest artifacts, as they are not lined by any form of epithelial cells as we should expect. The separating of the tubules and the appearance of the large intervening spaces in the sterile rats may be due to the shrinkage of the tubules, since the fibrous coat is puckered and the cells in most of the tubules show signs of degeneration; the cells, and especially the nuclei, do not stain well. There are some spermatozoa present, but the intratubular cells are smaller than those of young animals. Some of the tubules only show small lining cells and a mass of amorphous matter. The animals (2) which were looked upon as sterile had grown very large and the front teeth had grown so long that they seemed to have lost their usefulness; the hair was rough. They had, according to the dealer, been kept for a long time with females but showed no interest in them; this conclusion was supported by observations in this laboratory.
(b) Lipoids
In the embryo testes the interstitial cells which were mainly to be found in the centre of the organ contained lipoids (Fig. 6). The reason for the interstitial cells being more numerous in the centre is probably due to the tubules lying closer together at the periphery and leaving smaller interstitial spaces there. When the interstitial cells are seen near the margin they take the form of cords, whereas in the centre they are massed together. Large blood-vessels are present in the fibrous area at the centre of the organ.
At 2 weeks of age the interstitial cells which are found in the angles formed by the tubules contain lipoids, but from this stage to old age no lipoids are found in the comparatively few interstitial cells seen. During the 12th week lipoids are very prominent in some of the tubules; they are arranged inside the fibrous coat and between the cells, but not in the lumen as stated by Berberich and Jaffe and others. In the early stage of spermatogenesis there is a large amount of lipoid among the spermatogonia, and as spermatogenesis advances the amount becomes appreciably less until when free sperm is present in the tubules there is little or no lipoid present. This is well shown in Fig. 7. There is a difference in the affinity for the Sudan stain between the lipoids in the interstitial cells and those in the tubules. While the lipoids in the interstitial cells stain a faint orange colour and are diffuse those in the tubules are in the form of globules and are deeply stained and refractile.
SUMMARY
While in the mole, with its well-marked oestrous cycle, correlated changes occur in the cortex suprarenalis and in the interstitial cells of the testes, in the rat no change was found in the breadth or in the lipoid content of the cortex throughout the year.
The lipoids were evenly distributed throughout the cortex in the embryo, but from the age of 2 weeks onwards a lipoid free zone was present between the fasciculata and glomerulosa.
A detailed study of the histological development of the rat testes from embryonic life to old age showed that interstitial cells were proportionately more abundant in early life than in sexual maturity and old age.
Lipoids were present in the interstitial cells of the embryo and up to 2 weeks of life, after which age they were absent. In this respect the rat differs from the human subject, where the onset of puberty according to Mott (19) is accompanied by an increase in interstitial cells and the presence of lipoids in and around them.
The activity of spermatogenesis in the rat is enormous and it is possible that the lipoids of the interstitial cells are not allowed to accumulate, but are at once used up and that the cortex suprarenalis forms a reserve store. This seems to be indicated by the evidence from the mole.
From the 12th week lipoids appear between the cells lining the tubules of the testes ; these lipoids are most abundant in the tubules where spermatogenesis is at an early stage.
The results from the rat taken with those from the mole seem to indicate that in their storage of lipoids the cortical cells of the adrenals may be regarded as playing an ancillary part to the interstitial cells of the testes.
ACKNOWLEDGEMENTS
I am indebted to Professor Noël Paton for much kindly advice and encouragement.
My thanks are due to Mr J. R. Bell for the care he has taken in preparing the photographs.
The expenses of this research were defrayed from grants given by the Carnegie Trust and the Royal Society.
REFERENCES
PLATE XIV
Fig. 1. To show the granular cell layer between the glomerulosa and fasciculata in rat of 18 weeks, × 60.
Fig. 2. Section of suprarenal stained with Sudan III to show the lipoid free layer. Age 4 weeks, × 60.
Fig. 3. Section of testicle of 2 weeks embryo rat to show arrangement of tubules at periphery and large interstitial spaces towards centre, × 60.
Fig. 4. Section of testicle of old rat to show interstitial spaces and degeneration of tubules, × 60.
Fig. 5. Shows the inter-tubular spaces filled with fluid, the lymph spaces of Wagner. Age 12 weeks, × 350.