-

Spontaneous or Experimentally Induced Formation of a Special Zone in the Adrenal Cortex of the Adult Brush-Tailed Possum (Trichosurus vulpecula) J.B. KERR

AND

M. WEISS

Departments of Anatomy (J.B.K.) and Ph.ysiology (M.W.), Monash University, Clayton, Melbourne, Victoria, 3168 AustralLa

ABSTRACT The cytology and ultrastructure of the hypertrophied special zone, which is formed spontaneously in the adrenal cortex of adult female brush-tailed possums (Trichosurus vulpecula), was compared to the adrenocortical tissue in adult males in which the special zone, normally absent, was induced following castration alone or by additional treatment with folliclestimulating hormone (FSH). The special zone in females was situated between the zona fasciculata and the zona reticularis, the latter being a rudimentary zone in this species. Special zone tissue extended as a broad band parallel to and on one side of the adrenal medulla. In the luteal phase of the reproductive cycle, the special zone cells showed ultrastructural features commonly associated with steroidogenic tissues, with many mitochondria and compact masses of smooth endoplasmic reticulum. Cytoplasmic lipid inclusions were rarely observed. In lactating females, however, the special zone cells exhibited cytological and ultrastructural features suggestive of a transformation in their morphology broadly divided into two types of cells: (1) cells at the periphery of the special zone. close st^ to the zona fasciculata) showed variable quantities of lipid inclusions, mitochondria with dispersed cristae, and segregation of the smooth endoplasmic reticulum into compact masses; (2) cells within the more central regions showed an increasing abundance of lipid inclusions which in many cells became the dominant feature of the cytoplasm. These special zone cells contained very little smooth endoplasmic reticulum and their mitochondria contained few cristae together with amorphous granular material within the matrix. In castrated males, special zone tissue developed between the zona fasciculata and the zona reticularis, appearing initially as focal islands of cells (8 months postcastration) and later (11 months postcastration) expanding into a single zone, probably via the proliferation and differentiation of adjacent cells of the zona fasciculata and longitudinal growth of the special zone. Similar focal aggregations of special zone cells were in0 1991 WILEY-LISS, INC.

duced after 14 days of FSH treatment given to 2month castrated males. In all castrated and FSHtreated castrated males, the ultrastructure of special zone cells was similar to that of special zone cells in luteal-phase female possums. The findings suggest that the formation and cellular composition of the special zone is associated with changes in the pituitary-gonadal axis and that FSH plays a primary role in the differentiation of this tissue. INTRODUCTION

The adrenal glands of the brush-tailed possum (Trichosurus vulpecula) are unique among mammalian species in that the cortical tissue exhibits a distinct sexual dimorphism, first described by Bourne in 1934. In adult males the adrenal cortex is similar to the arrangement in placental species, displaying a n outer zona glomerulosa, a middle zona fasciculata, and a thin inner zona reticularis (Bourne, 1934, 1949; Chester Jones, 1957). In females, however, a n extra cortical zone is found on one side of the medulla, between the zona fasciculata and reticularis. This so-called special zone (Bourne 1934, 19491, although very small in young adult virgin females, may occupy 25-75% of’the volume of the cortex in older females (Bourne, 1949; Vinson et al., 1971; Call and Janssens, 1984). The special zone is undetectable in prepubertal animals of both sexes (Bourne, 1949; Chester Jones, 1957) suggesting that its postpubertal appearance is associated with hormonal changes during sexual maturation. Bourne’s studies in 1934 and 1949 were the first to investigate the cellular composition of the special zone based upon light microscopic examinations of paraffin sections obtained from museum specimens and from the wild population. Although the limited numbers of photomicrographs were difficult to interpret by modern standards, a t least three cell types (designated by Greek letters) were reported within the special zone. Deep to the zona fasciculata and forming the outer region of the special zone were the p cells characterized by a basophilic cytoplasm with little or no lipid inclu-

Received March 26, 1990.Accepted July 18, 1990. Address reprint requests to Dr. Jeffrey Kerr, Department of Anatomy, Monash University, Clayton, Melbourne, Victoria 3168, Australia.

J.B. KERR A N r) M. WEISS

102

sions. Chester Jones (1957) considered that the p cells were coextensive with the cells of the zona fasciculata. Bourne suggested that p cells transformed and proliferated into much larger eosinophilic 6 cells which often made up the major proportion of the special zone. A third cell type, the y cell, was occasionally scattered throughout the special zone, these cells being identified by a reduced amount of cytoplasm containing lipid inclusions. More recently, Call and Janssens (1984) described p and 6 cells in the special zone of adult female possums and questioned Bourne's (1949) suggestion that there were changes in special zone histology at particular stages of reproduction. However, representative micrographs of the proposed cell types were not available in their study, making it difficult to evaluate their conclusions. In adult females, 5a(P) reduced C21, C19 steroids are produced exclusively by the special zone tissue in contrast to the cortex proper which secretes A-4-3-keto hydroxysteroids such a s cortisol and corticosterone (Weiss and Ford, 1982, 1984; Edgar et al., 1989). It has been shown by Weiss and Carson (1987) that a clearly recognizable adrenocortical special zone can be induced in the male possum following short-term treatment with follicle-stimulating hormone (FSH) or long-term castration. Induction of special zone formation in adult males is accompanied by a change in the steroidogenic properties of the adrenal cortex in that the major products are 5a and 5p reduced steroids which are similar to those of adult females (Weiss and Carson, 1987). We were thus prompted in the present study to examine the cells of the special zone by electron microscopy and to compare the morphological features of experimentally induced special zone tissues of males with those of adult females. MATERIALS AND METHODS Animals and Treatments

Brush-tailed possums are a protected native species of Australia and permission was received from the Department of Conservation, Forests and Lands, State Government of Victoria, to obtain a limited number of animals for this study. Animals were wild-trapped in the sclerophyll forests surrounding Melbourne. Five adult females and five adult males were obtained and kept in a n animal house under controlled conditions on a diet of apples, carrots, and cabbage together with a mixed grain supplement. Of the females, two were in the luteal phase of the reproductive cycle and three were lactating with the ages of the pouch young ranging from 30-80 days. The stage of the reproductive cycle was determined using the criteria reported by Pilton and Sharman (1962). Four of the males were castrated via the scrota1 route under ketamine general anaesthesia (Ketalar; Parke, Davis & Co., Sydney; 0.5 mg/kg body weight via intramuscular injection). Two males, one a t 8 months and the other a t 11 months postcastration, were examined without additional treatment as described below. The remaining two animals a t 2 months postcastration received 14 daily i.m. injections of 2 mg porcine follicle-stimulating hormone (pFSH; NIH-FSH-P2, NIADDK, Baltimore, MD) which showed a biological potency of 0.69 NIH FSH S1 units per mg. The intact male possum was examined 2 months after capture.

Tissue Preparation far Light and Electron M~C~OSCOPY

Under ketamine general anesthesia, each animal received 5,000 units heparin via intraperitoneal injection. After 15 to 20 min of continued general anesthesia, the thoracic aorta was exposed and cleared of connective tissue and fat, following which a small incision was made in the vessel wall to permit the introduction of a polyethylene cannula of appropriate caliber. The cannula was connected to a 0.9% physiological saline solution a t room temperature with the addition of heparin (10,000 unitditer). When the cannula was secured into the aorta with surgical silk, the right atrium was severed and perfusion began under 150 cm of water pressure. Within 30-60 sec, the kidneys and adrenals became blanched; and the saline was replaced with fixative consisting of 3% glutaraldehyde, 2% formaldehyde, and 0.01% picric acid buffered in 0.1 M sodium cacodylate, pH 7.4. Perfusion continued for 45 to 60 min a t approximately 10 mls per min. The fixed adrenals were removed, cut into 1- to 2-mm slices along the sagittal or coronal planes, and immersion-fixed for a n additional 3 to 4 h r in the same fixative. All tissue slices were then cut into smaller blocks approximately 1-2 mm3, rinsed overnight in 0.1 M cacodylate buffer a t 4"C, and then postfixed for 3 h r in a buffered mixture of 1%osmium tetroxide and 1% potassium ferrocyanide. Tissues were thoroughly rinsed for 12 h r in cacodylate buffer and then stained en bloc for 2 hr in 1% uranyl acetate buffered with 0.05 M sodium maleate. Following several rinses in maleate buffer, blocks were dehydrated in a series of graded ethanol (70, 80, 90, loo%), cleared in two changes of propylene oxide, and embedded in a 1:l mixture of Ara1dite:Agar 100 (Agar Scientific, U.K.). Ovarian tissue from the luteal-phase female possums was fixed and processed as described above. Polymerized blocks were sectioned at 1 ym using glass or diamond knives, and sections were stained with 1%toluidine blue in 1%borax and examined and photographed by brightfield light microscopy. Areas selected for ultrastructural analysis were cut with a diamond knife; and sections of silver or gray interference color were collected on copper grids, stained with lead citrate and uranyl acetate, and examined with a Jeol 100s electron microscope operated a t 60 or 80 kV. Serial Sectioning of Adrenals

Adrenal glands from three other possums (one intact, adult female; one adult male castrated for 4 months; one adult male castrated for 2 months and given pFSH daily for 2 weeks) were cut in half along the coronal plane, fixed by immersion in Bouins solution, and embedded in paraffin wax. Each block was serially sectioned a t 6 pm parallel to the coronal plane, and every fourth section was mounted on a glass slide and stained with hematoxylin and eosin. From the 100 to 150 sections available from each block, every 10th or 12th section was analyzed by light microscopy; and the borders of the whole gland, the medulla, and the special zone (if present) were traced using a Zeiss MOP image analyzer (C. Zeiss, Oberkochen, W. Germany). The drawings were assembled a s a series of consecutive profiles, thus providing a macroscopic view of the relationship between the components of the adrenal tissue.

SPECIAL ZONE IN POSSUM ADRENAL CORTEX

103

Fig. 1. Low magnification of adrenal tissue of a lactating possum showing the position of the special zone (SZ)between the medulla {MI and the zona glomerulosa-zona fasciculata (ZG, ZF). Approximate boundary of the special zone is indicated by asterisks. x 65. Fig. 2. Arrangement of zona glomerulosa (ZG) and zona fasciculata (ZF) of adrenal cortex of a lactating possum. x 360.

RESULTS Adrenal Cortex of Females Light microscopy

The cortical tissue of the adrenal glands of all females contained an inner special zone of variable size but always positioned on one side of and adjacent to the medulla (Fig. 1). Although not surrounded by a sharp border or margin, the special zone could be readily determined by its less intense color with toluidine-blue staining and by a transitional band of tissue between it and the overlying zona fasciculata. At higher magnification the latter zone and the zona glomerulosa were characterized, respectively, by longitudinal columns of cells with moderate to plentiful supplies of lipid inclusions; and superficial to these, the cells of the zona glomerulosa showed increased basophilia and were arranged into arch-like configurations immediately deep to the capsule of the gland (Fig. 2). When cells of the special zone of luteal-phase possums were examined with high-resolution light microscopy ( x 63 oil-immersion objective), they showed a uniform morphology quite different from that of the zona fasciculata cells that surrounded the special zone (Fig. 3). The nuclei were ovoid or circular; and the cytoplasm contained many granules, although small areas of peripheral cytoplasm were also seen in which no granulation was

present. Most of the cells were 16-20 pm in diameter. An extensive network of sinusoidal-type blood vessels was observed. In contrast, special zone cells of lactating females exhibited a variety of morphologies that were 0; two major types. First, in the peripheral regions of the special zone, subjacent to the zona fasciculata, the special zone cells showed a gradual transition in structure in which those closest to the fasciculata contained a predominantly basophilic cytoplasm with granules, while cells slightly deeper into the special zone showed discrete aggregations of lipid inclusions together with patches of homogeneous basophilic cytoplasm (Fig. 4). Again many of the cells were 16-20 Fm in diameter. This pattern of cell morphology was also observed in the central region of the special zone of two of the lactating females whereas in the third animal, many of the cells exhibited remarkable accumulations of lipid droplets surrounding a small, deeply stained nucleus containing a small nucleolus and patches of heterochromatin (Fig. 5). These cells were often 30 pm or more in diameter. In order to compare the structure of adrenal cortex special zone cells with another typical steroidogenic tissue from the same animal, the corpus luteum was examined a t the same magnification. Luteal cells showed a very different morphology (Fig. 6 ) characterized by large nuclei often showing a single nucleolus and very little if any heterochromatin. The

Fig. 3. Cells of the special zone of a luteal-phase possum, showing arrangement of the cells into clumps and irregular anastomosing columns, separated by sinusoidal-type (S) blood vessels. Note patches of cytoplasm (arrows) devoid of granules. x 740.

Flg. 5. Cells in the central region of a special zone of a lactating possum, showing how each cell is filled with lipid inclusions (L), which surround the basophilic nuclei. A sinusoidal-type (S) blood vessel i s indicated. x 740.

Ftg. 6. Cells of the corpus luteum of a luted-phase possum, showing Fig. 4. Cells at the peripheral region of a special zone of a lactating possum, illustrating some cells partly filled with lipid inclusions (L) their cytoplasm filled with many small lipid inclusions and basophilic and others exhibiting a basophilic cytoplasm (C). A sinusoidal-type granules. A blood vessel (V) and fibroblasts (F) are shown. x 740. (S) blood vessel is shown. x 740.

SPECIAI, Z O N E I N POSSUM ADRENAL CORTEX

ri

SPECIAL ZONE

MEDULLA

Fig. 7. Three-dimensional reconstruction of the components of adrenal glands based upon assembly of paraffin sections cut in the coi-onal plane. Left, intact adult female; Middle, adult male castrated for 2 months and given pFSH daily for 2 weeks; Right, adult male castrated for 4 months.

cytoplasm of luteal cells was richly supplied with a variety of granules and inclusions, but no particular compartmentalization of these components was observed. The macroscopic features of the adrenal cortex special zone and the medulla are presented in Figure 7, illustrating the relatively large volume of the cortex occupied by the special zone. Electron microscopy

Cells from all zones of the adrenal cortex were analyzed by electron microscopy; and a n extensive description of the ultrastructure of cells from the zona glomerulosa, fasciculata, and the rudimentary reticularis is not presented here since their morphologies were comparable with numerous previous descriptions of these cells in other mammals. Cells of the zona glomerulosa showed irregularly shaped nuclei surrounded by a cytoplasm containing mitochondria with many tubular or lamellar cristae, and there were few lipid inclusions. Within the zona fasciculata, the chief structural change was the appearance of numerous lipid inclusions up to 2 pm in diameter with many mitochondria with tubular cristae interposed between the lipid inclusions. Zona reticularis cells were often fusiform in shape, their nuclei contained conspicuous patches of heterochromatin, and lipofuchsin pigments were observed in the cytoplasm. Occasionally nuclear pyknosis was seen; and macrophages were also present, their cytoplasm filled with lysosomes and electron-dense residual bodies. In confirmation of the observations of basophilic cells seen by light microscopy, special zone cells of lutealphase females showed a n abundance of mitochondria and peripheral areas of cytoplasai containing tubules of smooth endoplasmic reticulum (Fig. 8). Lipid inclusions were rarely observed. In lactating females, cells at the periphery of the special zone contained aggregations of lipid inclusions, patches of smooth endoplasmic reticulum, and mitochondria with tubular cristae together with one or more dense granules within the ma-

105

trix (Fig. 9). Another cell type in this region is shown in Figure 10 where the mitochondria exhibit a pale matrix and the cristae are dispersed giving a n empty appearance to the mitochondria. The smooth endoplasmic reticulum of these cells was more compressed into deeply stained areas bordered by dense membranes. Within the central area of the special zone of lactating females, the aforementioned cell types were observed; and in one animal most of the cells displayed small nuclei and the cytoplasm was now almost filled with lipid inclusions (Fig. 11). High magnification of the cytoplasm revealed relatively few organelles between the many lipid inclusions. Mitochondria exhibited a n unusual morphology characterized by very few, short, lamellar cristae; and the matrix was partly filled with amorphous granular material (Fig. 12). Membranes of smooth or rough endoplasmic reticulum were noted between the lipid inclusions. When these cells were compared with similar lipid-rich cells of the zona fasciculata from the same adrenals, a very different morphology was observed; in the latter the cytoplasmic matrix contained many mitochondria with tubular cristae together with abundant supplies of tubules of smooth endoplasmic reticulum (Fig. 13). Adrenal Cortex of Males Light microscopy

The adrenal cortex of the untreated adult male possum showed a conventional histological arrangement similar to that of placental species (Fig. 14). In castrated males, a new zone of cortical tissue appeared between the zona reticularis and the zona fasciculata; and this special zone tissue occurred either in focal aggregations (Fig. 15) or as a single large special zone on one side of the medulla (Fig. 16).Similar focal areas of special zone cells were also observed after FSH treatment (Fig. 17). Two histological patterns of organization of the special zone were displayed. In the first, special zone cells appeared to merge or were continuous with the adjacent zona fasciculata (11-month castrated and FSH treatment); and in the second arrangement the special zone was flanked by a thin border of elongated cells (&month castrated). The unique morphology of special zone cells was evident with higher magnification light microscopy when they were compared to cells of the cortex proper. Zona glomerulosa cells contained small amounts of lipid inclusions; whereas in the outermost regions of the zona fasciculata, the cells were filled with lipid occupying much of their cytoplasm (Fig. 18).Within the deeper or innermost regions of the zona fasciculata, the cells were arranged into longitudinal columns; but they showed few if any cytoplasmic lipid inclusions (Fig. 19). Cells of the zona glomerulosa and fasciculata were usually between 14 and 20 pm in diameter. In the special zones of ll month castrated males, the cells were ovoid or irregularly elongated in shape, they were much larger than other cortical cells (up to 35 pm in largest diameter), and their cytoplasm was moderately basophilic and showed many pale or dark granules, but lipid inclusions were not identified (Fig. 20). Toward the periphery of the special zone, adjacent to the zona fasciculata but not to the zona reticularis, it was common to see mitotic figures in which the nuclear membrane had disappeared and one or more chromosomes

Fig. 8 . Ultrastructure of a typical special zone cell o f a luteal-phase possum, showing the nucleus (N) and aggregations of smooth endoThe mitochondria (M) contain both lamellar plasmic reticulum 6). and tubular cristae. X 7500. Fig. 9. Ultrastructure of a special zone cell similar to those shown in Figure 4 showing the nucleus (N), smooth endoplasmic reticulum

( S ) , lipid inclusions (L), and mitochondria (MI displaying tubular

cristae and the matrix often containing one or more granules. X 7100. Fig. 10. Ultrastructure of a special zone cell in a peripheral region of a special zone of a lactating possum, showing aggregations of smooth endoplasmic reticulum (S) often bordered by dense membranes (arrows). The mitochondria (M) exhibit a pale matrix and dispersed cristae. Lipid inclusions (L) are abundant. x 7800.

Fig. 11. Ultrastructure of cells within the central region of a special zone of a lactating possum showing the cytoplasm filled with lipid inclusions which surround centrally or peripherally placed nuclei (N). x 1900. Fig. 12. Ultrastructural detail of the cytoplasm ofspecial zone cell of a lactating possum similar to Figure 11. Between the numerous lipid inclusions (L), the mitochondria (M) shuw very few cristae and the

matrix contains dense amorphous material. Tubules of smooth endoplasmic reticulum ( 5 )and vesicles of rough endoplasmic reticulum (R) are shown. x 20,000. Fig. 13. Ultrastructure of a typical zona fasciculata cell of a lactating possum (similar to Fig. 2) for comparison with Figure 11.Between the lipid inclusions (L),the cytoplasm contains many mitochondria (M) showing a conventional morphology. x 7400.

Fig. 14. Low magnification of adrenal tissue of a normal male possum, illustrating the capsule ( c ) ,zona glomerulosa (ZG), zona fasciculata (ZF) and medulla (M). Zona reticularis is not readily identified at this power. x 130.

Fig. 15. An island of special zone cells (SZ) in the adrenal cortex of an 8-month castrated possum, showing the boundary tissue surrounding the zone. x 140.

Fig. 16. Adrenal tissue of an 1 1-month castrated possum, illustrating a very large special zone (SZ) whose margins are indicated by the asterisks. X 55.

Fig. 17. Cortex of’adrenal gland of an FSH-treated castrated possum showing a reginn of special zone cells (SZ), which merges into the overlying area of zona fasciculata (ZF). x 105.

Fig. 18. Light micrograph of adrenal cortex of a normal male possum showing a thin layer of zona glomerulosa iZG) and the underlying irregular columns of zona fasciculata cells with abundant cytoplasmic lipid inclusions. A sinusoidal-type (S) capillary is shown. x 780. Fig. 19. Light micrograph of the inner region of zona fasciculata cells showing thcir basophilic cytoplasm. Note sinusoidal-type ( S )capillaries. x 780.

Fig. 20. Light micrograph of a central region of a special zone of a n 11-month castrated possum, illustrating arrangement of cells into irregular cords separated by sinusoidal-type is) capillaries. x 850. Fig. 21. Light micrograph ofthe peripheral region of a special zone of an 11-month castrated possum, illustrating several cells (arrows) containing chromosomes indicating cell division. x 850.

110

J.B. KE:RR AND M. WEISS

were visible (Fig. 21). The location and relative size of the special zone in castrated male possums is indicated in Figure 7.

types. In agreement with Bourne (1949), the special zone exhibits a n asymmetrical location on one side of the medulla; and i t often shows a characteristic pale appearance when stained with toluidine blue. Bourne Electron microscopy reported that the special zone consisted of fi, 6, and y Ultrastructural analysis of special zone cells from cell types, the former two types predominating and the castrated males revealed the presence of dark and light latter a n inconstant feature. Similar observations were cells with a variety of shapes, sizes, and form and con- made by Call and Janssens (1984), although no refertent of cytoplasmic organelles and inclusions (Fig. 22). ence was made to the y cells. Our observations indicate In the 8-month castrated male, some special zone cells three cell types each with a characteristic morphology, contained many mitochondria with very unusual mor- possibly forming a spectrum of morphological features phologies (Fig. 23). Often the mitochondria appeared reflecting changes in cell maturation and function. We grossly swollen, and the few small cristae present were have found it difficult to compare our observations of positioned along the inner membrane bordering the special zone cell morphology with those published 40 large areas of matrix. These cells also contained small years ago by Bourne (19491, and we cannot interpret aggregations of smooth endoplasmic reticulum, the the data by Call and Janssens (1984) since photomicroGolgi apparatus was prominent, and lysosomes were graphs were not included in their report. The relatively numerous. Another type of special zone cell from the poor definition of cell structure in Bourne’s work does same animal showed moderate electron density due to not permit a comparison of the cytoplasmic features of large compacted masses of smooth endoplasmic reticu- the various special zone cells, a criterion of key signiflum, usually positioned towards the periphery of the icance in ascertaining the existence of the three cell cell (Fig. 24). The remainder of the cytoplasm con- types. Our findings allow us only to state that the three tained scattered smooth membranes, lysosomes, and cell types range morphologically from those not convery small lipid inclusions; and again the mitochondria taining lipid to others showing accumulation of lipid showed a central matrix devoid of cristae, the latter together with segregation of smooth endoplasmic reticoften positioned adjacent to the inner membrane. ulum and mitochondria with very few cristae, and fiIn the 11-month castrated male, the special zone nally to a third cell type with a condensed nucleus and cells exhibited a similar ultrastructure; but the smooth filled with remarkable quantities of lipid inclusions endoplasmic reticulum appeared more compact, being and mitochondria almost depleted of their cristae. We surrounded and interdigitated by parallel stacks of can confirm Bourne’s (1949) suggestion of considerable smooth membranes (Fig. 25). The mitochondria exhib- fluctuations in the proportions of different cell types in ited lamellar and tubular cristae, although the central the special zone of lactating females, since with differmatrical areas often lacked cristae. Thus in castrated ent age pouch young, i t was noted in one lactating female possums, the ultrastructural features of the in- male that the majority of special zone cells contained duced special zone tissue resemble those of the adult abundant lipid, whereas in two others the special zone female in which the special zone is always present. The cells showed less lipid and thus resembled cells with a n principal difference between the male and female spe- intermediate morphology between entirely basophilic cial zone morphology is the accumulation of lipid in- cells (no lipids) and very weakly stained cells (rich supclusions in lactating females. Special zone cells of the plies of lipid). Whether or not these differences reprecastrated males treated with FSH for 14 days showed sent variations in morphology due to cell maturation or all the features expected of a n accredited steroidogenic changes responding to the reproductive status of inditissue, although there was less ultrastructural vari- vidual animals can only be answered by future examability between the cells compared to special zone cells ination of greater numbers of specimens. The concept from long-term castrated males. In most cells the mi- that the proportions of p and 6 cells vary a t different tochondria were numerous and contained many tubu- stages of the reproductive cycle was questioned by Call lar cristae; Golgi membranes and lysosomes were and Janssens (1984) who failed to detect any cyclic patnoted, and the cytoplasm contained abundant smooth tern in cell numbers or types. They concluded that it endoplasmic reticulum which was often compacted into was unlikely that the special zone was involved in or large aggregations towards the periphery of the cell responded to any specific reproductive function. How(Fig. 26). ever, when their data on the relative areas occupied by p and 6 cells within the special zone are recalculated, DISCUSSION the results support the hypothesis of a transition from This is the first study to examine the ultrastructure p to 6 cells since the differences attained a significant of the adrenal cortex of the brush-tailed possum with statistical difference (P < 0.05, t-test) not reported in emphasis upon the morphology of the special zone the study. which is unique to this species. The observations show The interrelationship between special zone cells and that the induced or naturally occurring special zone their unique morphology raises the question of the orconsists of cells with a variety of ultrastructural fea- igin of these cells. They must arise either from the zona tures, all of which are commonly associated with a ste- fasciculata or the zona reticularis. Bourne (1949) sugroidogenic function. gested that growth of the special zone might be achieved by maturation of cells near the medulla and Special Zone in Females outward expansion, implying a reticularis origin for We have confirmed that the inner adrenal cortical the special zone. We suggest the opposite, i.e., developtissue of the adult female possum contains a large hy- ment from the surrounding fasciculata zone, for the pertrophied special zone consisting of a number of cell following reasons. Recent 3H-thymidine labeling stud-

SPECIAL ZONE: IN POSSUM AI)IIENAI, CORTEX

Fig. 22. Ultrastructural features of special zone cells of a n 8-month castrated possum illustrating the variety of dark and light cells that are supplied by a network of capillaries ( C ) . x 2600.

111

112

J . H . KERR AND M. WEISS

Fig, 23. Ultrastructure of a special zone cell of a n 8-month castrated possum illustrating mitochondria (M) many with clear matrices due t o a paucity of cristae. Regions of smooth endoplasmic reticulum (S), Golgi membranes ( G ) ,and numerous lysosomes (Ly) are present. x 7500.

SPECIAL ZONE I N POSSUM ADRENAT, CORTEX

Fig. 24. Ultrastructure of special zone cells of an 8-month castrated possum. The cells show areas of dark and light density due respectively to aggregations of compact membranes of smooth endoplasmic reticulum (CS) and to a cytoplasmic matrix exhibiting numerous mitochondria (M) often containing few cristae. Interdigitation (arrows) of adjacent plasma membranes is shown. x 4900.

113

114

.J.B. KERR AND M. WEISS

Fig. 25.Ultrastructure of a typical special zone cell of a n 11-month castrated possum, showing aggregations of smooth endoplasmic reticulum ( S ) associated with wave-like cytoplasmic densities (arrows). Numerous mitochondria (MI are shown. x 7500. Inset: Higher magnification of aggregated smooth endoplasmic reticulum showing dense parallel arrays of membranes. x 17,300.

ies of the rat adrenal cortex by Zajicek et al. (1986) have provided evidence favoring the ‘cell migration theory’ (Gottschau, 1883) in which adrenocytes from the superficial cortex migrate into the deeper cortex, expressing a characteristic morphology and steroidogenic capacity when traversing each zone. This theory differs from the ‘zonal theory’ (Chester Jones, 1948), which regards the three zones as separately active and self-renewing (reviewed by Idelman, 1978). The cell-migration concept is compatible with the notion that transformation of cells from the deep zona fasciculata would provide p cells a t the periphery of the special zone, which, under appropriate stimuli (or withdrawal of particular factors), ‘metamorphose’ into the 6 cells. Although our study was not designed to consider the question of the kinetics of special zone proliferation, two pieces of circumstantial evidence support the above hypothesis; (1) zona fasciculata cells gradually merge with cells of the special zone (Figs. 1,3,4),and (2) the transitional cell type in lactating females (containing lipid and smooth endoplasmic reticulum components) was located between the zona fasciculata and the special zone proper (Fig. 4),but these cells were not observed adjacent to the zona reticularis. The occasional presence of a thin band of attenuated cells be-

tween the induced special zone and the zona fasciculata in castrated males (Fig. 15) raises the additional possibility that the growth of the focal islands of special zone cells is achieved via longitudinal expansion as suggested by the serial-section reconstructions. Clearly the kinetic properties and developmental history of the special zone must await further study with 3H-thymidine techniques to trace the growth and displacement of cells within the whole adrenal cortex. Ultrastructural examination of special zone cells revealed a number of alterations in morphology that were not seen in the remainder of the adrenal cortex: (1) increasing abundance of lipid as the predominant feature of the cytoplasm; (2) sequestration of smooth endoplasmic reticulum into compact masses and eventual reduction in amount; (3) accumulation of intramitochondria1 granules and significant loss of cristae; (4)reduction in nuclear size. These observations suggest a process of marked metabolic alteration a n d o r cell degeneration, yet the cells become progressively larger rather than smaller as would be expected if degeneration was their fate. We found no evidence of cell pyknosis except in the cells of the zona reticularis; and, in addition, the macrophages located here often contained phagocytosed cellular debris, but this did not

SPECIAL ZONE IN POSSUM ADRENAL CORTEX

Fig. 26. Ultrastructure of a special zone cell of an FSH-treated castrated possum, illustrating membranes of smooth endoplasmic reticulum ( S )often aggregated into compact areas of smooth membranes (CS). Mitochondria (M) contain tubular cristae. Dense lysosomes (Ly) and occasional vesicles of rough cndoplasmic reticulum (R)are shown. X 12,000.

115

116

J.R. KERR AND M. WEISS

occur within the special zone itself. Special zone cells filled with lipid inclusions and containing abnormal mitochondria thus resemble zona fasciculata cells in the hypophysectomized rat in which the cells undergo ‘fatty degeneration’, i.e., they show condensation of the nucleus, increased amounts of lipid, and few cristae in the mitochondria (Fujita, 1972). When steroidsecreting cells accumulate many lipid inclusions, it is generally believed that their overall steroidogenic capacity is either changed andlor reduced (Christensen and Gillim, 1969; Idelman, 1970, 1978; Nussdorfer et al., 1978). In placental species, the adrenal cortex also accumulates numerous lipid inclusions following experimental withdrawal of corticotropin (ACTH) or administration of selective metabolic or steroidogenic enzyme inhibitors (Idelman, 1978).It is well documented that the special zone tissue, in addition to its content of hydroxylating enzymes, contains very active 5a and 5P reductases, which is in contrast to the cells of the cortex proper (Weiss and Ford, 1982; Edgar et al., 1989).Furthermore, the possibility that variations in special zone ultrastructure are accompanied by changes in steroidogenic properties is supported by the in vitro study of Weiss (1985) who showed that the activities of the special zone reducing enzymes varied both quantitatively and qualitatively in association with the female reproductive state. In that study the total yield of 5aand 5P-reduced steroids produced from progesterone substrate was reduced in pregnant or estradiol-treated females compared to untreated virgin females. Special Zone in Males and its Relationship to Females

The present study shows that it is possible to induce special zone formation in male possums by either longterm (4,8, or 11 months) castration or by administration of FSH following a shorter period (2 months) of castration. Whereas the development of the special zone is slow in castrated males, it could be greatly enhanced by treatment with FSH. These findings extend previous work describing FSH-induction of special zones in castrated males in which the tissues were identified only by light microscopy of paraffin sections (Weiss and Carson, 1987). FSH binding sites were found on both the adrenal cortex proper and special zone of females (Risbridger and Weiss, 1985). Thus the ‘anlagen’ for special zone formation is present in both sexes, and it is obvious that the male is a more suitable model for the study of the origin and growth of the special zone, as well as the cell transformation process suggested by Bourne (1949). The length of the postcastration period seemed to influence the size of the special zone, which was largest and extended as a continuous band along one side of the medulla in the 11month castrated male (Fig. 16). In the 8-month castrated male, a number of islets of special zone cells appeared in different sites, again adjacent to the medulla, and a similar pattern of special zone development was noted for FSH-treated males. In both castrated and castrated plus FSH-treated males, special zone cells exhibited ultrastructural features indicative of a steroidogenic function, although in the castratedalone animals a greater variety of cellular organelles and inclusions was seen. The appearance of a special zone in the male adrenal cortex is accompanied by a change in the pattern of steroidogenesis which becomes

similar to that of the special zone in females, i.e., synthesis of mainly 5P-reduced steroids (Weiss and Carson, 1987). Another well-known cortical zone is the adrenal fetal zone in primates, but it disappears soon after birth (Bloch,1968).The so-called X zone of the mouse adrenal cortex (Masui and Tamura, 1926; Howard-Miller, 1928) consists of acidophilic cells that encircle the medulla, and the cells vary in abundance according to sex and age (Idelman, 1978). The X zone is recognizable in immature mice of both sexes whereas in the brush-tailed possum males of all ages lack a special zone. In postpubertal male mice, however, the X zone degenerates and atrophies as a result of increasing androgen secretion by the developing testes (Deansley and Parkes, 1937; Chester Jones, 1949a; Holmes and Dickson, 1971). If immature male mice are castrated, the X zone persists (Howard, 1939); and when adult males were castrated, a secondary X zone was formed from the inner zona fasciculata (Chester Jones, 1949b, 1955) and this is very similar t o special zone formation in castrated male possums. In both of these studies LH (but not FSH) was implicated as the stimulating agent. In contrast to the cells of the special zone, X zone cells are smaller than zona fasciculata cells (Ross, 1967; Sato, 1967; Hirokawa and Ishikawa, 1974); and although their ultrastructure is typical for a steroidogenic tissue (Sato, 1967,19681, they share few of the unusual morphological features seen in special zone cells of male or female possums. So far, the only enzyme system detected in the X zone is a 20a-hydroxysteroid dehydrogenase (Ungar and Stabler, 1980). The variable morphology of special zone cells in luteal-phase or lactating possums and the induction of a special zone in castrated or FSH-treated male possums suggest that changes in reproductive status influence the function of the adrenal cortex. Previous studies of the relationship between the pituitary-gonadal axis and the adrenal cortex in male and female possums has established a major role for FSH in stimulating special zone formation (Weiss and Carson, 1987). The spontaneous or experimentally induced development of the special zone raises questions about the physiological significance of this tissue. It is clear from the ultrastructural studies presented here that special zone cells show all the morphological characteristics of steroidogenic cells, and many of the cells appeared structurally similar in males and females. One exception was the appearance of many lipid inclusions in special zone cells of one lactating female, a feature not seen in the male adrenal. Whether these particular cells secrete steroids different from those produced by cells not containing lipid inclusions (e.g., luteal-phase females and males) can be determined only by analysis of the steroidogenic capacity of isolated subpopulations of special zone cells. The overall secretory products of male or female special zone tissue are known to be C19 and C21, 5a-and 5P-reduced steroids (Weiss and Ford, 1982; Weiss, 1984; Weiss and Carson, 1987). These unique steroids are known to modulate brain function and to inhibit uterine contraction in eutherian species (Kubli-Garfias et al., 1976, 1979, 1980). Since these reduced steroid products are found in high concentrations in the adrenal venous blood of female possums

SPECIAL ZONE I N POSSUM ADKENAT, CORTEX

(Edgar et al., 19891, it seems likely that there are extra-adrenal target tissues capable of responding to these steroids. In conclusion, the observations indicate that special zone tissue can be induced in the adrenal cortex of male possums. The cell ultrastructure is similar to that of the special zone cells, which occur spontaneously in all adult female possums. The growth of the special zone is influenced by the pituitary-gonadal axis, and the available evidence shows that FSH has a primary role in the function of the adrenal cortex of this species. A larger number of animals at different stages of the reproductive cycle and post-partum will need to be investigated to establish if the characteristics of special zone morphology are associated with specific secretory functions of physiological importance. ACKNOWLEDGMENTS

This work was supported by the Australian Research Grants Scheme (No. A187158931 and a Monash University Special Research Grant (No. 19.186.028).Animals were obtained with the permission of the Department of Conservation, Forests and Lands, State Government of Victoria (Permit No. 88-022).We thank C.M. Knell and A. Martsi for technical assistance. LITERATURE CITED Bloch, E. 1968 Fetal adrenal cortex. In: Functions of the Adrenal Cortex, Vol. 2. K.W. McKerns, ed. North-Holland, Amsterdam, pp. 723-750. Bourne, G.H. 1934 Unique structure in the adrenal of the female possum. Nature, 1342t664-665. Bourne, G.H. 1949 The Mammalian Adrenal Cortex. Oxford University Press, London. Call, R.N., and P.A. Janssens 1984 Hypertrophied adrenocortical tissue of the Australian brush-tailed possum (Trzchosurus uulpeculal: Uniformity during reproduction. J. Endocrinol., 1012t263267. Chester-Jones, I. 1948 Variation in the mouse adrenal cortex with special reference to the zona reticularis and to brown degeneration, together with a discussion of the “cell migration” theory. Q.J. Micros. Sci., 89t53-74. Chester Jones, I. 1949a The action of testosterone on the adrenal cortex of the hypophysectomized, prepubertally castrated male mouse. Endocrinology, 44:427-438. Chester Jones, I. 1949b The relationship of the mouse adrenal cortex to the pituitary. Endocrinology, 45.514-536. Chester Jones, I. 1955 Role of the adrenal cortex in reproduction. Br. Med Bull., 11:166-160. Chester Jones, I. 1957 The Adrenal Cortex. Clarendon Press, Oxford. Christensen, A.K., and S.W. Gillim 1969 The correlation of fine structure and function in steroid secreting cells with emphask on those of the gonads. In: The Gonads. K.W. McKerns, ed. NorthHolland, Amsterdam, pp. 415-488. Deansley, R., and A.S. Parkes 1937 Multiple activities of androgenic compounds. Q.J. Exp. Physiol., 26r393-402. Edgar. J.A., M. Weiss, and K.A. Than 1989 Identification of 5p-pegnane and 5P-androstane derivatives in adrenal venous and peripheral blood plasma of the female possum (Trichosurus uulpeculal. J. Steroid Biochem., 322t56.5-572. Fyjita, H. 1972 Fine structure of alterations of the adrenal cortex in hypophysectomized rats. Z. Zellforsch., 125:480-496. Gottschau, M. 1883 Struktur und embryonale entwickelung der

117

nebennieren bei saugetieren. Arch. Anat. Entwwgesch., 9:412458. Hirokawa, N., and H. Ishikawa 1974 d e c t r o n microscopic observations on postnatal development of the X zone in the mouse adrenal cortex. Z. Anat. Entwgesch., 1442t85-100. Holmes, P.V., and A.D. Dickson 1971 X-zone degeneration in the adrenal glands of adult and immature female mice. J. Anat., 108: 159-168. Howard, E. 1939 Effects of castration on the seminal vesicles as influenced by age, considered in relation to the degree of dcvelopment of the adrenal X-zone. Am. J. Anat., 65.105-149, Howard-Miller, E. 1928 A transitory zone in the adrenal cortex which shows age and sex relationships. Am. J. Anat., 40r251-293. Idelman, S . 1970 Ultrastructure of the mammalian adrenal cortex. Int. Rev. Cytology, 27:181-281. Idelman, S.1978 The structure of the mammalian adrenal cortex. In: General, Comparative and Clinical Endocrinology of the Adrenal Cortex. I. Vol. 2. Chester Jones and I.W. Henderson, eds. Academic Press, New York, pp. 1-199. Kubli-Garfias, C., M. Cervantes, and G. Beyer 1976 Changes in multiunit activity and EEG induced by the administration of natural progestins to falxedil immobilized cats. Brain Res., 114t71-81. Kubli-Garfias, C., L. Medrano-Conde, G . Beyer, and A. Bondani 1979 In vitro inhibition of rat uterine contractility induced by 5u and 5p progestins. Steroids, 34509-617. Kubli-Garfias, C., A. Lopez-Fiesco, M. Pacheco-Cano, H. Ponce Monter, and A. Bondani 1980 In vitro effects of androgens upon the spontaneous rat uterine contractility. Steroids, 351633-641. Masui, K., and Y. Tamura 1926 The effects of gonadectomy on the structure of the suprarenal gland of mice with reference to the functional relationship between this gland and the sex glands of the females. J. Coll. Argic. Imp. Univ. Tokyo, 7:353-376. Nussdorfer, G.G., G . Mazzocchi, and V. Meneghelli 1978 Cytophysiology of adrenal zona fasciculata. Int. Rev. Cytology, 55:291-365. Pilton, P.E., and G.B.Sharman 1962 Reproduction in the marsupial (‘Trichosurus Z J U ~ ~ ~ C U J. ~ UEndocrinol., ). 25:119-136. Risbridger, G.P., and M. Weiss 1985 Gonadotrophin and steroid binding to adrenal cortex tissue of female possum f Trichosurus uulpecula). Aust. J. Zool., 33:831-835. Ross, M.H. 1967 Fine structure of the juxtamedullary region of the mouse adrenal cortex with special reference to the X-zone. Anat. Rec., 157t313. Sato, R. 1967 Age and sex differences in the fine structure of the mouse adrenal cortex. Nagoya J. Med. Sci., 30r225-251. Sato, T. 1968 The fine structure of the mouse adrenal X-zone. Z. Zellforsch., 87:315-329. Ungar, F., and T.A. Stabler 1980 20o-hydroxysteroid dehydrogenase activity and the X-zone of the female mouse adrenal. J. Steroid Biochem., 1323-28. Vinson, G.P., J.G. Phillips, I. Chester Jones, and W.N. Tsang 1971 Functional zonation of adrenocortical tissue in the brush possum (Trichosurus uulpeculal. J. Endocrinol., 49:131-140. Weiss, M. 1984 Gonadotrophin induced development of the special zone in the adrenal cortex of the immature possum (Trichosurus uulpecula) with concomitant activation of steroid reductases. Comp. Biochem. Physiol., 79B:173-179. Weiss, M. 1985 Factors influencing adrenocortical special zone steroidogenesis in the possum (Trichosurus uulpecula). 10th Int. Symp., Comp. Endocrinol., Colorado, U.S.A., Abst. 96. Weiss, M., and R.S. Carson 1987 Induction of adrenocortical special zone in the male possum (Trichosurus uulpecula). Comp. Biochem. Physiol., 86A:361-365. Weiss, M., and V.L. Ford 1982 Sex differences in steroidogenesis by adrenal homogenates of the adult possum (Trichosurus 0~1pt.cula) attributable to the steroids formed by the adrenocortical special zone of the female. Gen. Comp. Enducrinol., 462t168-175. Weiss, M., and V.L. Ford 1984 Differences in steroidogenevis by subcellular fractions of adrenocortical special zone and cortex proper of the female possum (Trichosurus uulpeculaj. J . Steroid Biuchem., 21t701-707. Zajicek, G., I. Ariel, and N. Arber 1986 The streaming adrenal cortex: direct evidence of centripetal migration of adrenocytes by estimation of cell turnover rate. J. Endocrinol., lllt477-482.