Cell Tissue Res. 196, 23-38 (1979) Cell and Tissue Research �9 by Springer-Verlag 1979

Three Dimensional Organization of Mammalian Adrenal Cortex

A Scanning Electron Microscopic Study

Pietro Motta*, Masaki Muto and Tsuneo Fujita

Department of Anatomy, Faculty of Medicine, University of Rome, Italy Department of Anatomy, Niigata University School of Medicine, Niigata, Japan

Summary. The adrenal cortex of different mammals was studied by SEM in order to demonstrate its actual three-dimensional organization. In the rat, as well as in the cat and pig, the adrenal cortex appeared as a "tunnelled continuum" of polyhedral cells arranged in plate-like structures (laminae). This laminar arrangement was more evident in the inner fasciculate and reticular zones where the cortex revealed a striking similarity to liver tissue. The polyhedral cells of all cortical zones possessed regular facets populated by small pits, larger invaginations and numerous microvilli with the exception of very short and smooth areas probably corresponding to attachment zones and/or gap junctions. This cellular architecture produced a labyrinthic system of intercellular channels or lacunae in which the capillaries were suspended.

The pericapillary areas of this labyrinth contained microvilli, amorphous material, a delicate net of fibrils and occasional cells. The intercellular compartment of this lacunar system was mainly bordered by numerous microvilli arising from endocrine cells.

The luminal surface of the capillary wall showed not only irregularly protruding margins (interpretable as endothelial junctions) but also clearly overlapping and flattened endothelial extensions.

In all the animals and areas of the adrenal cortex examined, the endothelial wall was provided with abundant clusters of small fenestrations (about 50 nm in diameter) generally arranged in sieve plates.

Larger fenestrations were noted mainly in the fasciculate and reticular zones of the cat and pig and occasionally in the rat.

A final point related to the nature and significance of sinusoidal fenestrations was the occurrence of irregularly shaped and intracapillary located cells mainly noted in the deeper zones of the fasciculate and reticular zones of the gland. These elements - possessing the surface characteristics of macrophages - were observed, with their irregular and slender evaginations, in

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24 P. Motta et al.

close proximity to the large fenestrations in a manner reminiscent o f Kupffer cells within the lumen of liver sinusoids.

Key words: Adrenal cortex - Mammals - Three-dimensional organizat ion - Scanning electron microscopy.

The fine structure o f the adrenal cortex has been extensively studied in different labora tory mammals and some h u m a n specimens under normal and experimental condit ions (Zelander, 1959; Cotte and Cotte, 1961; Long and Jones, 1967; Idelman, 1970; Rhodin, 1971; Kurosumi and Fujita, 1974; Malamed, 1975). Despite all these studies knowledge o f the three-dimensional architecture o f this endocrine organ is based only on diagrams obtained f rom models reconstructed f rom serial histological sections (Elias and Pauly, 1956; Pauly, 1957).

On the other hand, some questions posed by early light microscopists (Bourne, 1949) such as the nature o f capillaries and their morphological and functional relationship to the cortical endocrine cells, have been variously interpreted in the light o f transmission electron microscope (TEM) investigations (Zelander, 1964; Luse, 1967; Idelman, 1970; Rhodin, 1971).

The present SEM study aims to demonstra te the three-dimensional organi- zat ion o f the adrenal cortex o f different mammals and to provide detailed informat ion relevant to the surface characteristics o f the epithelial cells and the structure o f the capillaries and associated intercellular and pericapillary spaces.

Materials and Methods

Adrenal glands of 5 adult normal rats, two cats and one pig were used. The animals were anesthetized with an intramuscular or intraperitoneal injection of Nembutal (0.1 ml/kg/animal) and perfused through the aorta (pig) and/or the aorta or the left ventricle (rats and cats) with heparinized Tyrode solution or Ringer solution at room temperature or gently warmed (37~ Gravity maintained a perfusion flow of about 60 ml/min. After about 30-50 s the flow of the solution was interrupted and 2.5 % of glutaraldehyde buffered with sodium phosphate (0.1 M; pH 7.4) or cacodylate (0.18 M; pH 7.4) was perfused for about 1 0-15 rain. After perfusion the adrenals were excised and immersed in the same fixative for a few days and then, reduced in smaller blocks, were treated with the tannin-osmium impregnation method of Murakami (1974). The fragments were then washed several times in distilled water and dehydrated in increasing concentrations of ethanol and transferred to isoamylacetate. After the blocks were freeze-cracked in liquid nitrogen (Tokunaga et al., 1974) and critical point-dried in liquid CO 2 using a JEM apparatus, the fragments mounted on metal stubs using conducting silver paint were coated with gold palladium in a vacuum evaporator (type HUS-4 Hitachi) under continuous rotation and tilting of the specimens. All the material was studied and photographed in a field emission type SEM (HFS-2 Hitachi) operated at 10 kV.

Results

In all the animals studied, the zonat ion o f the cortical areas was apparent, and made more obvious due to the ar rangement and course o f the blood vessels (Fig. 1).

These vessels, (mainly capillaries), observed in longitudinal or transversal fractures, possessed a rather variable diameter which ranges between 8 and 16 ~tm, and a number o f interconnections or anastomoses (Fig. 2).

SEM of Mammalian Adrenal Cortex 25

Fig. 1. Survey of dissociated adrenal cortex. C fibrose capsule; G zona glomerulosa; Fzona fasciculata; R zona reticularis; M medullary region. (Cat). x 290

Fig. 2. Survey of a fractured adrenal cortex. G zona glomerulosa; Fzona fasciculata; R zona reticularis. Large vessels are evident in the medullary area (M). Characteristic is the arrangement of the capillary net in the cortex. (Rat). x 320

26 P. Motta et al.

The capillaries thus assumed the aspect of a network of dilated channels corresponding to the sinusoidal type of capillaries described in the liver (Figs. 2, 3). Furthermore, in the deeper zones, the capillaries appeared highly enlarged and entered wide collecting vessels (veins) located in the center of the medulla (Figs. 2, 3).

In some instances, larger vessels (likely arteries) appeared to run directly from the capsule to the medulla without ramifications (Fig. 3).

Generally, the capillaries of the cortex showed a more obvious and tortuous course, and lateral sacculations, in addition to their progressively larger caliber, passed from the glomerulosa to the fasciculata and from the latter to the reticularis (Figs. 1-3).

In all the animals observed, the capillary wall consisted only of very flattened endothelial cells. Their luminal surface was directly bulging only in the region of the cell that contained the nucleus (Fig. 4). Cell boundaries were easily noted and the cellular attachments appeared roughly protruding and sometimes, provided with a few, short, microvilli (Figs. 4, 5). In other locations the thin endothelial expansions appeared overlapping (Figs. 5, 6).

The luminal surface of the capillary showed a number of irregularly shaped or flattened protrusions of cytoplasm (Figs. 4, 7 and an occasional single cilium (Fig. 8). More characteristic of the endothelial wall however, was the presence of numerous fenestrations (Figs. 4-7). In all the animals, and in all the different cortical areas examined, the fenestrations usually appeared as rounded and oval pores measuring 50-100 nm in diameter. They rarely occurred in isolation but as a rule, grouped in clusters, often forming "sieve plates". The cluster arrangement of the pores was more frequently observed in the rat capillaries (Figs. 4, 7). In the pig (Fig. 5), and cat (Fig. 6) the pore clusters were variable in size. Furthermore, other types of endothelial gaps were occasionally noted in these animals, and especially, in the inner fasciculate and reticular zones. These were larger than the pores described above and showed a variable diameter (more than 100 nm) and a round or oval shape (Fig. 9).

The capillary lumen of the pig adrenal in the areas of the inner fasciculate and reticular zones was occasionally occupied by solitary and irregularly shaped cells (Figs. 9-11). These elements possessed an irregularly elongated cell body and a variable number of superficial cell extensions. At higher magnification the surface of both cell body and cytoplasmic protrusions appeared rough due to the presence of a number of small invaginations, blebs, ruffles and short microvilli. In some cases the cellular extensions of these cells assumed the shape of lamellipodia and filopodia (Figs. 9-11).

These intraluminally located cells with their flattened and slender ends then appeared either overlapping the endothelial wall in areas provided with large fenestrations, or penetrating through these gaps, assumed a relationship with the subjacent pericapillary space. Mainly in the same areas where the above cells were encountered, other irregularly shaped cell protrusions were noted to emerge from the subcapillary spaces and project into the capillary lumen through other gaps (Figs. 9-11).

In all the cases observed, and in every zone of the adrenal cortex, a pericapillary space was always recognized. This space was variable in width (0.2-0.6 Ilm) and was

Fig. 3. Zona reticularis (R) in which are evident numerous capillaries penetrating (arrows) large vessels (veins) present in the medulla (M). A larger channel (likely an arteriole) (A) cross the cortical zone and opens into a large medullary vein directly (arrow plus asterisk). (Pig). x 500

Fig. 4. A cellular body likely containing the nucleus (N) is evident in this picture. From it arises a number of large cellular protrusions similar to arms (arrows) with interconnected flattened endothelial expansions in which are evident numerous pores ("sieve plates"). (Zona fasciculata of rat). x 14,000

28 P. Motta et al.

Figs. 5 and 6. Overlapping endothelial cells (arrows). Note the fenestrated nature of the sinusoidal wall provided with small and large gaps. A few fibers lying in the subendothelial spaces are evident through the larger fenestrations. Dense spheroidal bodies of different size and unknown nature are scattered into the capillary lumen (asterisks). A bulbous protrusion is also evident emerging through a fenestration of the sinusoidal wall (asterisk plus arrow). (Fig. 5: Transitional zone between fasciculata and reticularis of pig; x 24,000) (Fig. 6: Zona fasciculata of cat; x 49,000)

Fig. 7. Endothelial wall showing irregularly protruding arms and flattened extensions largely fenestrated ("sieve plates"). A cortical cell faces the endothelial wall into the pericapillary space with numerous microvilli (m). (Zona reticularis of rat). x 35,000

Fig. 8. Endothelial lumen showing a few isolated cilia (arrows). An irregular cell body (macrophage?) likely contained into a pericapillary space is evident (Pc). (Zona reticularis of pig). x 5,400

Fig. 9. Irregularly shaped cell is present within the sinusoidal lumen (M). The cytoplasmic extensions of this element (likely corresponding to a macrophage) are related to large endothelial fenestrations (arrows). Other large gaps are also evident in adjacent areas (F). (Zona reticularis of pig). • 12,500

Fig. 10. Stereo view of sinusoids and cortical cells. Note the continuous extensions of the labyrinthic system of pericapillary and intercellular lacunae in which the microvilli of the cortical cells are projected. In the upper part of the micrograph is present an histiocyteqike cell located into the sinusoidal lumen. This element is provided with large cellular extensions contacting some fenestrations. (Zona reticularis of pig). x 4,000

30 P. Motta et al.

Fig. 11. Survey of the laminar-like arrangement of the zona reticularis. The wide intercellular and perisinusoidal spaces (arrows) are occupied by numerous microvilli of the endocrine cells. The capillary lumen (S) is occupied in two cases by irregularly shaped cells likely representing macrophages (M). (Pig). x 2,700

SEM of Mammalian Adrenal Cortex 31

Fig. 12. A number of fibers 00 and dense spheroidal bodies of different sizes are present in the pericapillary spaces and in the sinusoidal lumen. Microvilli and small invaginations are present on the cell surfaces. The endothelial wall is provided with small fenestrations arranged in clusters (c) and some large gaps (F). (Zona fasciculata of cat). x 17,000

Fig. 13. Survey of the three-dimensional aspect of the zona glomerulosa. E endocrine cells; S sinusoids. (Rat). x 1,400

Fig. 14. Polyhedral cell with angular facets. Microvilli (m) and a few invaginations are scattered on these surfaces. (Zona glomerulosa of cat). x 8,000

32 P. Motta et al.

larger and more irregularly shaped mainly in the inner fasciculate and reticular zones than in the zona glomerulosa (Figs. 10, 11). This space was clearly occupied by a different amount of fine fibrillar and amorphous material likely corresponding to the reticular fibers associated to the basal lamina (Fig. 12).

The pericapillary space was always populated by numerous microvilli arising from the cortical cells and by a number of dense spheroidal bodies of different size similar to other structures observed within the capillary lumen (Fig. 12). As observed in panoramic views and stereo pairs (Figs. 10, 11) the pericapillary space was continuous with irregularly shaped recesses or intercellular spaces which actually, as microchannels bordered by microvilli, connected adjacent pericapillary spaces. Thus a three-dimensional net of intercellular and pericapillary lacunae was formed.

Occasionally profiles of irregularly shaped and pericapillary located cells were also noted in different zones (Fig. 8).

Cortical Cells

SEM observations showed that the cortex of the adrenal glands was composed of continuously interconnected groups of irregularly polyhedral cells separated from the capillary walls by a wide lacunar space (Figs. 1, 2).

The zone glomerulosa was composed of relatively arch-like and compacted groups of interconnected cells which together assume a cordon-like or laminar-like aspect (Fig. 13). The glomerulosa cells possessed an irregular, angular or elongated shape and an average width of about 10-15 lam. Small invaginations (pits), short microvilli and spherical bodies of different size were observed on the facets of the glomerulosa cells facing the intercellular and subendothelial spaces (Fig. 14).

The zonafasciculata consisted of polyhedral cells which were larger than those of the glomerular zone (with an average width of 15-20 I~m) and which were arranged in sparsely interconnected laminae (Fig. 15). The facets of these cells frequently appeared well delineated and showed a concave shape provided with long microvilli projecting into the intercellular and pericapillary spaces (Figs. 16, 17). Small invaginations and a number of spheroidal bodies were distributed on the surface of these cells (Figs. 15-17).

The zona reticularis was made up of polyhedral cells (average width of 10-15 ~tm) which formed a three-dimensional system of interconnected irregular plate-like structures (Fig. 18). Also, in this case, the cellular facets were populated by a variable number of microvilli, invaginations (pits) and spheroidal dense bodies of unknown nature (Fig. 19).

Fig. 15. Laminar arrangement of cells (E) and associated sinusoids (S) of the zona fasciculata. The fractured cells show empty cavities likely corresponding to the cytoplasmic areas containing lipid droplets and/or other cell organelles (lysosomes and mitochondria). (Rat). x 1,900

SEM of Mammalian Adrenal Cortex 33

Fig. 16. Fractured endocrine cell of the zona fasciculata. The concave facet shows long microvilli (m) and a few invaginations (arrows). Small spheroidal and dense bodies of unknown nature populate these areas and in some location (asterisk plus arrows) are in close relation with the cortical invaginations. (Cat). x 21,500

Fig. 17. High magnification of cell surfaces facing intercellular (/) and/or pericapillary lacunae (P). These areas are populated by microvilli and dense spheroidal bodies of unknown nature. The cell surfaces possess a number of small invaginations (pits) (arrows) in relation with minute bodies (1 plus arrow). (Rat). x 19,600

34 P. Motta et al.

Fig. 18. Survey of the zona reticularis. The sinusoidal net (S) and the laminar-like arrangement of the cortical cells (E) are patent. In some regions are also evident intracapillary and/or pericapillary located cells (histiocytes?) (arrows). Wide intercellular and perisinusoidal spaces containing microvilli are shown in several places (Pig). x 1,400

Fig. 19. Endocrine cell surfaces provided with microviUi (m) and numerous cortical invaginations (arrows) are evident in this picture. En endothelial wall with a different population of gaps. D dense bodies of unknown nature (Zona reticularis of cat). x 8,600

SEM of Mammalian Adrenal Cortex 35

Discussion

Histological investigations based on models obtained from reconstructed serial sections of rat and human adrenal glands showed that the cortex was a "tunnelled continuum" of cells arranged in interconnected groups (Elias and Pauly, 1956; Pauly, 1957).

The present SEM investigation readily confirms this assumption and extends the observations to different species of mammals. Furthermore, the findings showed that in the rat, as well as in the pig and cat, the structure of the three cortical zones was a tunnelled continuum of polyhedral cells associated with each other to form not cords but, as in the liver, mainly plate-like structures (laminae). This laminar arrangement in the adrenal cortex was more evident in the inner fasciculate and reticular zones where the similarity with the hepatic parenchyme as revealed by recent SEM studies was very striking (Motta and Porter, 1974; Grisham et al., 1976). Utilizing stereoviews and selected and magnified areas of adrenal cortex, it was noted that the epithelial cells possessed rather regular facets provided with microvilli. Only some short areas of these facets were closely attached to the neighboring cells likely by means of junctional zones. This cellular arrangement produced a labyrinthic system of intercellular channels or lacunae in which the capillaries were actually suspensed. The pericapillary areas of this three- dimensional labyrinth of lacunae contained microvilli, an amorphous material (likely the basal lamina) and a delicate net of fibrils into which occasional pericapillary cells were noted. On the contrary, the intercellular compartment of this lacunar system was mainly bordered by the microvilli arising from the cortical cells.

As a result of this close relationship between cortical cells and capillaries, it was obvious under the SEM that almost each single facet of the epithelial cells was directly or indirectly related to the endothelial wall. Therefore, the SEM observations not only confirmed the real existence of these spaces as reported in a number of previous TEM investigations (Lever, 1956; Cotte and Cotte, 1961; Zelander, 1964; Long and Jones, 1967; Luse, 1967; Idelman, 1970; Rhodin, 1971) but, by adding a three-dimensional evaluation, clearly demonstrates that these spaces are actually part of a continuous lacunar system which permeates all the cell and endothelial surfaces. Probably as in other endocrine organs, this system of microchannels, which runs along the microvillous surfaces of the epithelial cells and the endothelial wall can be interpreted as a means for facilitating the transport of secretory product from the cells to the blood stream.

Obviously, in the adrenal gland - as likely in other endocrine tissues - this lacunar system can also be involved in the transport of nutrients from the blood to the endocrine cells and/or be a site where both blood and cellular filtrates (fluids) are temporarily accumulated before they might reach the lymphatic vessels. In this regard, the fine structural organization of the adrenal cortex seems to be similar to that of the liver (Motta, 1977a). Both organs in fact possess similar, large, pericapillary and intercellular spaces, and both liver and adrenal are apparently devoid of typical lymphatical capillaries. Thus in the liver these spaces ("Disse's spaces") are regarded by physiologists as special lymphatic lacunae in which the tissues and blood filtrates flow from the central part of the lobule to the portal spaces of Mall and lymphatic vessels (Brauer, 1963). Similarly, in the adrenal

36 P. Motta et al.

cortex, fluids might flow from these spaces into the lymphatic vessels located within the connective tissue of the capsule or around the large medullary vessels (Harrison, 1960).

In regard to the vascularization of the cat, rat and pig adrenal gland, the present SEM investigation basically provided similar information. If the three-dimensional arrangement of capillaries is regarded with particular reference to their irregular course, variable caliber and association with the endocrine cells, there is no doubt that they can be termed "sinusoids" (Long and Jones, 1967). Further, other SEM details have demonstrated on the luminal surface of the sinusoidal wall, irregularly protruding margins interpretable as attachment zones between adjacent cells. These SEM results confirm previous TEM observations (Idelman, 1970; Rhodin, 1971) and are similar to endothelial junctions noted in other capillaries and/or vessels using SEM methods (Smith et al., 1971; Tokunaga et al., 1973; Peine and Low, 1975).

In other locations, flattened endothelial extensions of sinusoids appear to overlap. In particular, overlapping endothelial cells have been rarely described in the adrenal cortex sinusoids, and were reported only in a photograph by Luse (1967), and mentioned, but denied, by Bloodworth and Powers (1968). The frequent occurrence of overlapping endothelial cells reported in this paper can be explained with the fact that the use of the SEM permitted the analysis of larger areas of the luminal sinusoidal wall at one time.

SEM observations also showed that the endothelial wall, at the time of fixation, was provided with numerous clusters of small fenestrations (pores) having a rather constant diameter of 50--100 nm. Due to the actual resolution power of the SEM instrument, it was not possible to decide whether these pores were provided with a real diaphragm as clearly reported in other TEM investigations (Long and Jones, 1967; Bloodworth Jr. and Power, 1968; Rhodin, 1971). In addition the present study demonstrated that open fenestrations were evident in other areas of the endothelial wall. Open fenestrations in the adrenal sinusoids larger than 50 m~t, were reported for different mammalian species (Lever, 1956; Zelander, 1959; Sheridan and Belt, 1964; Brenner, 1966; Long and Jones, 1967; Luse, 1967; Idelman, 1970; Porter and Bonneville, 1973; Kurosumi and Fujita, 1974). In the present study, openings of the capillary wall were noted mainly in the fasciculate and reticular zones of the cat and pig and occasionally, in the rat. Small fenestrations or pores were observed in all the above species, and in all the areas of the gland. Although it is possible to consider that the type and number of fenestrations might be related to a species difference and/or to the nutritional state of the animal, it cannot be excluded that the larger openings might be artifactually generated. As in the case of the liver (Wisse, 1972; Brooks and Haggis, 1973; Itoshima et al., 1974; Motta and Porter, 1974; Grisham et al., 1975; Motta, 1975; Muto, 1975; Motta et al., 1978) it is plausible to consider that the sinusoidal wall is very delicate, then it is not difficult to imagine that the larger fenestrations could be temporary, possibly even formed by a process of coalescence of the smaller ones (Grisham et al., 1976), as a response to various physiopathological events (Motta et al., 1978).

A final point which is indirectly related to the nature and significance of sinusoidal fenestrations is the occurrence of irregularly shaped cells which were located in intracapillary regions. In fact, considering both the shape and the location in the sinusoidal lumen of these cells (mainly in close proximity to the

SEM of Mammalian Adrenal Cortex 37

larger fenest ra t ions) it seems reasonable to conclude tha t the presence o f such large gaps might be dependen t u p o n an ac t iva t ion o f these cells (Mot t a , 1977a, b).

As observed under the SEM, these cells closely resembled mac rophages descr ibed in a n u m b e r o f recent S E M studies o f o the r o rgans (Car r and Carr , 1970; Fu j i t a et al., 1972; A lb rech t et al., 1972; H o s o y a and Fuj i ta , 1973; Mot t a , 1975; M u t o , 1975, 1976; M o t t a et al., 1977). Therefore if these cells co r r e spond to mac rophages it is logical to assume tha t they actual ly are the same macrophages or his t iocytes descr ibed bo th in the per is inusoidal spaces (Zelander , 1964; Long and Jones, 1967; Rhod in , 1971) and into the capi l la ry lumen ( Ide lman, 1970).

On the o ther hand , it is also poss ible to suggest tha t they might be ac t iva ted monocy tes present wi th in the s inusoidal lumen and in the process o f pene t ra t ing the subendothe l ia l areas t h rough the large gaps. I f these conclus ions are correct , it is per t inent to suggest tha t the mac rophages o f the adrena l gland, with the s inusoidal wall to which they are re la ted can be included in the "mononuclear phagocyte system" which was recent ly p r o p o s e d to replace (see: B loom and Fawcet t , 1975) the " reticuloendothelial system of Aschoff'.

Acknowledgements. This paper is dedicated to the memory of Professor W. Bargmann because we considered the criticisms and suggestions he provided for this article as probably the last ones before his death.

The authors are pleased to acknowledge the excellent technical assistance of Mr. K. Adachi. Thanks are also due to Dr. J. VanBlerkom for his kind revision and criticism of the manuscript. The work was supported by funds from the National Council of Researchs (CNR) of Italy and from the Japan Society for Promotion of Sciences (JSPS).

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Accepted September 25, 1978