Alteration of Subcapsular Adrenocortical Zonation in Humans with Aging: The Progenitor Zone Predominates over the Previously Well-Developed Zona Glomerulosa after 40 Years of Age

Few studies have examined functional adrenal zonation throughout human life. Adrenals from 61 surgical/autopsy patients from 1 day old to 92 years old who had no clinical endocrinological/mineralocorticoid abnormalities were assessed for immunohistochemically defined adrenal zonation. The zona glomerulosa (zG) was well developed in all 11 patients ranging in age from newborn to the 30s. After 40 years of age, however, the zG occupied less than one-quarter of the adrenal circumference, suggestive of zG involution. The other subcapsular areas were occupied by the progenitor zone (zP), which expressed neither cytochrome P450_aldo nor P450_11β but 3β-hydroxysteroid dehydrogenase and P450scc, although some autopsy cases had adrenals with zG zonation because of secondary aldosteronism, and others who had experienced severe stresses showed subcapsular zona fasciculata (zF). In conclusion, the adrenal cortex consists of homogeneous zG-topped columns from birth to adolescence. Subsequently, in the fifth decade of life, the cortex is reconstituted by integration of three types of cortical columns: scattered zG-topped columns and zonal zP-topped columns, the latter having the ability for bidirectional differentiation into either zG-topped columns or zF-topped columns, according to secondary aldosteronism or the presence of severe stresses. Such adrenocortical remodeling is ascribed to high-sodium/low-potassium diets.     

Ascorbate stimulates monooxygenase-dependent steroidogenesis in adrenal zona glomerulosa

It is well known that ascorbic acid (Asc) is highly concentrated in the adrenal gland, but its function in the gland is not thoroughly elucidated. We therefore examined the possibility that Asc participates in steroidogenic monooxygenase systems of the adrenal cortex with the aid of the regenerating system including outer mitochondrial membrane cytochrome b (OMb). When Asc availability was limited in rat mutants unable to synthesize Asc, the increase in plasma aldosterone concentration under Na-deficiency was suppressed without effect on plasma corticosterone concentration. Aldosterone formation in the isolated mitochondrial fraction of the zona glomerulosa (zG) of the adrenal cortex was stimulated by the addition of Asc and NADH, while corticosterone formation was not. Consistently zG showed a high level of Asc regeneration activity and was rich in OMb among adrenocortical zones. Taken together, the enhanced aldosterone formation that is catalyzed by one of the steroidogenic monooxygenases, P450aldo, may be supported by Asc with its regenerating system.     

Conditionally immortalized adrenocortical cell lines at undifferentiated states exhibit inducible expression of glucocorticoid-synthesizing genes.

To facilitate studies on differentiation of adrenocortical cells and regulation of steroidogenic genes, we established cell lines from adrenals of adult transgenic mice harboring a temperature-sensitive large T-antigen gene of simian virus 40. Adrenal glands of the mice exhibited normal cortical zonation including a functionally undifferentiated cell-layer between the aldosterone-synthesizing zona glomerulosa cells and the corticosterone-synthesizing zona fasciculata cells. At a permissive temperature (33 degrees C), established cell lines AcA201, AcE60 and AcA101 expressed steroidogenic genes encoding steroidogenic factor-1, cholesterol side-chain cleavage P450scc, and steroidogenic acute regulatory protein, which are expressed throughout adrenal cortices and gonads. Genes encoding 3 beta-hydroxysteroid dehydrogenase and steroid 21-hydroxylase P450c21, which catalyze the intermediate steps for syntheses of both aldosterone and corticosterone, were inducible in the three cell lines in temperature- and/or dibutyryl cAMP-dependent manners. Notably, these cell lines displayed distinct expression patterns of the steroid 11 beta-hydroxylase P45011 beta gene responsible for the zone-specific synthesis of corticosterone. AcA201 cells expressed the P45011 beta gene at 33 degrees C, showing the property of the zona fasciculata cells, while AcE60 cells expressed it upon a shift to a nonpermissive temperature (39 degrees C). On the other hand, AcA101 expressed the P45011 beta gene at 39 degrees C synergistically with exposure to dibutyryl cAMP. None of these clones express the zona glomerulosa-specific aldosterone synthase P450aldo gene under the conditions we tested. These results show that AcE60 and AcA101 cells display a pattern of the steroidogenic gene expression similar to that of the undifferentiated cell-layer and are capable of differentiating into the zona fasciculata-like cells in vitro.     

Morphology and ultrastructure of the adrenal gland in Bactrian camels (Camelus bactrianus)

In the present study, we examined the morphological features of the adrenal gland in Bactrian camel by means of digital anatomy, light and electron microscopy. Our findings testified that the gland was divided into three parts, capsule, cortex and medulla from outside to inside as other mammals, and the cortex itself was further distinguished into four zones: zona glomerulosa, zona intermedia, zona fasciculate and zona reticularis. Notably, the zona intermedia could be seen clearly in the glands from females and castrated males, whereas it was not morphologically clear in male. There was a great deal of lipid droplets in the zona fasciculate, while it was fewer in the zona glomerulosa and zona reticularis. The cytoplasm of adrenocortical cell contained rich mitochondria and endoplasmic reticulum. The adrenal medulla was well-developed with two separations of external and internal zones. The most obvious histological property of adrenal medulla cells were that they contained a huge number of electron-dense granules enveloped by the membrane, and so medulla cells could be divided into norepinephrine cells and epinephrine cells. Moreover, the cortical cuffs were frequently present in adrenal gland. Results of this study provides a theoretical basis necessary for ongoing investigations on Bactrian camels and their good adaptability in arid and semi-arid circumstances.     

Aldosterone biosynthesis in mitochondria of isolated zones of adrenal cortex

An assumption that the aldosterone-synthesizing enzyme exists only in zona glomerulosa cells apparently contradicts our recent findings that a purified bovine adrenocortical cytochrome P-45011 beta catalyzes the aldosterone formation and the enzyme exists in both zones of the adrenal cortex. To gain more insight into the zone specificity of aldosterone production, the aldosterone-synthesizing activity of mitochondria prepared from the isolated zones of adrenal cortex of various animal species was investigated. The intact mitochondria from the bovine or porcine zonae fasciculata-reticularis could not produce aldosterone whereas those from the zona glomerulosa produced it at a significant rate. When the mitochondria from the zonae fasciculata-reticularis were solubilized by the addition of cholate, they produced aldosterone from corticosterone at a rate comparable to that of those from the zona glomerulosa. The presence of specific factor(s) in the zonae fasciculata-reticularis mitochondria inhibiting expression of the aldosterone synthetic activity is discussed. The mitochondria of the rat zonae fasciculata-reticularis could hardly catalyze aldosterone synthesis under the detergent-solubilized conditions, whereas those of the zona glomerulosa could. Immunoblot analysis revealed that the mitochondria of the zonae fasciculata-reticularis contained a protein of Mr 51,000 which was immunocrossreactive with a monoclonal antibody directed against P-45011 beta, whereas those of the zona glomerulosa contained two immunocrossreactive proteins of Mr 51,000 and 49,000. These results suggest that in the case of rat adrenal cortex, a specific aldosterone-synthesizing enzyme exists in the zona glomerulosa.     

Studies on cytogenesis in adult rat adrenal cortex: circadian and zonal variations and their modulation by adrenocorticotropic hormone

Circadian rhythms and zonal variations in the cell proliferation of adult rat adrenal cortex were studied by following the cells in the DNA-synthesizing stage (S-phase) as assessed by 5-bromo-2'-deoxyuridine incorporation into the cell-nuclei and/or by visualizing proliferating cell nuclear antigen. The S-phase cells were observed throughout the day in two regions of the adrenal cortex: (i) a region from the inner half of the zona glomerulosa to near the outer margin of the zona fasciculata, and (ii) the outer one-fourth portion of the zona fasciculata. Very little change in number was observed in the former region between day and night, while a burst of cell proliferation occurred in early morning at 3-4 a.m. in the latter region. A prominent rise in the plasma adrenocorticotropic hormone (ACTH) concentration preceded the burst of cell proliferation by about 4 h. Upon raising the plasma ACTH concentration by administration of ACTH or metyrapone, prominent cell proliferation also occurred in the same portion of the zona fasciculata 4-6 h after the provoked ACTH surge. Thus at least two sites in rat adrenal cortex are responsible for cytogenesis in this endocrine organ, and respond differentially to day/night cycles and circulating ACTH levels.     

Synthetic modified N-POMC1–28 controls in vivo proliferation and blocks apoptosis in rat adrenal cortex

The identity of the pro-opiomelanocortin (POMC)-derived mitogen in the adrenal cortex has been historically controversial. We have used well-established in vivo models, viz., hypophysectomized (Hyp) or dexamethasone (Dex)-treated rats, to study the effect of the synthetic modified peptide N-terminal POMC (N-POMC_1–28) on DNA synthesis in the adrenal cortex, as assessed by BrdU incorporation and compared with adrenocorticotropic hormone (ACTH). We evaluated the importance of disulfide bridges on proliferation by employing N-POMC_1–28 without disulfide bridges and with methionines replacing cysteines. Acute administration of synthetic modified N-POMC_1–28 distinctly increased DNA synthesis in the zona glomerulosa and zona fasciculata, but not in the zona reticularis in Hyp rats, whereas in Dex-treated rats, this peptide was effective in all adrenal zones. ACTH administration led to an increase of BrdU-positive cells in all adrenal zones irrespective of the depletion of Hyp or Dex-POMC peptides. The use of the ACTH antagonist, ACTH_7–38, confirmed the direct participation of ACTH in proliferation. Two different approaches to measure apoptosis revealed that both peptides similarly exerted a protective effect on all adrenocortical zones, blocking the apoptotic cell death induced by hypophysectomy. Thus, ACTH_1–39 and N-POMC_1–28 have similar actions suggesting that the disulfide bridges are important but not essential. Both peptides seem to be important factors determining adrenocortical cell survival throughout the adrenal cortex, reinforcing the idea that each zone can be renewed from within itself.     

Strain differences in rabbit adrenocortical structure and morphological evidence of corticomedullary communication

Interstrain differences in the structure of the mouse and rat adrenal cortex are well known, but related data on rabbits are not available. This study was planned to demonstrate possible strain differences in rabbit adrenal morphology and morphological evidence of a communication between cortex and medulla. For this purpose, the zonation of the adrenal cortex of intact mature male and female rabbits of different strains (New Zealand, Chinchilla and California) were compared using morphometry and the corticomedullary junction was evaluated for close relationship. Marked intersex and interstrain differences were found in the adrenocortical zonation. Female rabbits had larger adrenocortical zones than corresponding males. The rabbits of Chinchilla and California strains had the largest cortical sizes. This difference depended largely upon the higher thickness of the zona fasciculata. Whereas, there were no significant strain differences in the thickness of zona glomerulosa and zona reticularis. Furthermore, examination of corticomedullary junction showed that cortex and medulla were interwoven. Single and small accumulations of cortical cells were spread throughout the medulla. Our morphological data showed the presence of significant interstrain differences in the adrenocortical zonation in the rabbits and provide evidence for a possible paracrine interaction between medullary and cortical cells.     

Immunohistochemical localization of adrenal ferredoxin and distribution of adrenal ferredoxin and cytochrome P-450 in the rat adrenal

Adrenal ferredoxin, the iron-sulfur protein associated with cytochromes P-450 in adrenocortical mitochondria, has been localized immunohistochemically at the light microscopic level in rat adrenals by employing rabbit antiserum to bovine adrenal ferredoxin in both an unlabeled antibody peroxidase-antiperoxidase method and an indirect fluorescent antibody method. When sections of rat adrenals were exposed to the adrenal ferredoxin antiserum in both procedures, positive staining for adrenal ferredoxin was observed in parenchymal cells of the three cortical zones but not in medullary chromaffin cells. Marked differences in the intensity of staining, however, where observed among the three cortical zones: the most intense staining being found in the zona fasciculata, less in the zona reticularis, and least in the zona glomerulosa. Furthermore, differences in staining intensity were also observed among cells within both the zona fasciculata and the zona reticularis. In agreement with these immunohistochemical observations, determinations of adrenal ferredoxin contents by electron paramagnetic resonance (EPR) spectrometry in homogenates prepared from capsular and decapsulated rat adrenals revealed that the concentration of adrenal ferredoxin in the zona glomerulosa was lower than that in the zona fasciculata-reticularis. Similar results were obtained when the contents of cytochrome P-450 were determined in capsular adn decapsulated rat adrenal homogenates. These observations indicate that adrenal ferrodoxin and cytochrome P-450 are not distributed uniformly throughout the rat adrenal cortex.     

Proliferation and distribution of adrenocortical cells in the gland of ACTH- or dexamethasone-treated rats

The effects of prolonged (7-day) ACTH and dexamethasone administrations on rat adrenocortical-cell turnover have been investigated by combined stereological and metaphase-arrest techniques. ACTH was found to increase the number of parenchymal cells in each adrenal zone; however, ACTH altered the cell distribution in the cortex, lowering their percentage in the zona glomerulosa (ZG) and zona fasciculata (ZF) and enhancing it in the zona reticularis (ZR). The cell birth-rate was markedly raised by ACTH exclusively in ZG and ZF. Dexamethasone notably decreased the number of ZF and ZR cells, without altering that of ZG cells. Moreover, dexamethasone increased the percentage of parenchymal cells in ZG and ZF, and lowered it in ZR. In the adrenal cortices of dexamethasone-administered animals, metaphases were virtually absent. These data indicate that ACTH increases the cell birthrate in ZG and possibly ZF, and enhances the centripetal migration of newly-formed cells and their accumulation in ZR. Dexamethasone inhibits both proliferation of adrenocortical cells in the outer cortical layers and their centripetal migration into ZR. Moreover, it appears to cause parenchymal-cell loss in the inner adrenocortical layers.     

Morphological evidence for a close interaction of chromaffin cells with cortical cells within the adrenal gland

Summary The adrenal medulla appears to exert a regulatory influence on adrenocortical steroidogenesis. We have therefore studied the morphology of rat, porcine and bovine adrenals in order to characterize the contact zones of adrenomedullary and adrenocortical tissues. The distribution of chromaffin cells located within the adrenal cortex and of cortical cells located within the adrenal medulla was investigated. Chromaffin cells were characterized by immunostaining for synaptophysin and chromogranin A, both being considered specific for neuroendocrine cells. Cortical cells were characterized by immunostaining for 17-hydroxylase, an enzyme of the steroid pathway. Cellular contacts of chromaffin cells and cortical cells were examined at the electron microscopical level. In rat and porcine adrenals, rays of chromaffin cells, small cell clusters and single chromaffin cells or small invaginations from the medulla could be detected in all three zones of the cortex. Chromaffin cells often spread in the subcapsular space of the zona glomerulosa. In porcine and bovine adrenals, 17-hydroxylase immunoreactive cells were localized within the medulla. Single cortical cells and small accumulations of cells were spread throughout this region. At the ultrastructural level, the chromaffin cells located within the cortex in pig and rat adrenals formed close cellular contacts with cortical cells in all three zones. Our morphological data provide evidence for a possible paracrine role of chromaffin cells; this may be important for the neuroregulation of the adrenal cortex.     

Immunohistochemical studies on electron transport proteins associated with cytochromes P-450 in steroidogenic tissues. II. Microsomal NADPH-cytochrome c reductase in the rat adrenal.

NADPH-cytochrome c reductase (NADPH : ferricytochrome oxido-reductase, EC 1.6.2.4), the flavoprotein which mediates the NADPH-dependent reduction of cytochromes P-450 in adrenocortical microsomes, has been localized immunohistochemically at the light microscopic level in rat adrenal glands. Localization was achieved through the use of sheep antiserum produced against purified, trypsin-solubilized rat hepatic microsomal NADPH-cytochrome c reductase in both an unlabeled antibody peroxidase-antiperoxidase technique and an indirect fluorescent antibody method. The sheep antibody to rat hepatic microsomal NADPH-cytochrome c reductase concomitantly inhibited the NADPH-cytochrome c reductase and progesterone 21-hydroxylase activities catalyzed by isolated rat adrenal microsomes. When sections of rat adrenal glands were exposed to the reductase antiserum in both immunohistochemical procedures, positive staining for NADPH-cytochrome c reductase was observed in parenchymal cells of the three cortical zones but not in medullary chromaffin cells. The intensity of staining, however, was found to differ among the three cortical zones, with the most intense staining being found in the zona fasciculata and the least in the zona glomerulosa. The intensity of staining was also found to differ among cells within the zona fasciculata. These immunohistochemical observations demonstrate that microsomal NADPH-cytochrome c reductase is not distributed uniformly throughout the rat adrenal cortex.     

Relationship between mitochondrial lipid peroxidation and alpha-tocopherol levels in the guinea-pig adrenal cortex

Lipid peroxidation in mitochondria from the functionally distinct inner (zona reticularis) and outer (zona fasciculata + zona glomerulosa) zones of the guinea-pig adrenal cortex was investigated. Ferrous ion (Fe2+)-induced lipid peroxidation was far greater in inner than outer zone mitochondria. Ascorbic acid similarly initiated lipid peroxidation to a greater extent in inner zone mitochondrial preparations. Differences in the unsaturated fatty acid content of inner and outer zone mitochondria could not account for the regional differences in lipid peroxidation. Total fatty acid concentrations were greater in the outer than in the inner zone, and the relative amounts of each fatty acid were similar in the two zones. However, mitochondrial concentrations of alpha-tocopherol, an antioxidant known to inhibit lipid peroxidation, were approx. 5-times greater in the outer than inner zone. The results demonstrate that there are regional differences in mitochondrial lipid peroxidation in the adrenal cortex which may be attributable to differences in alpha-tocopherol content. Thus, alpha-tocopherol may serve to protect outer zone mitochondrial enzymes from the consequences of lipid peroxidation and thereby contribute to some of the functional differences between the zones of the adrenal cortex.     

Modulation of the effects of ascorbic acid on lipid peroxidation by tocopherol in adrenocortical mitochondria

Studies were done to evaluate the role of alpha-tocopherol in modulating the effects of ascorbic acid (AA) on lipid peroxidation (LP) by adrenocortical mitochondria. In control mitochondria from the inner (zona reticularis) or outer (zona fasciculata plus zona glomerulosa) zones of the guinea pig adrenal cortex, subphysiological concentrations of AA stimulated LP but higher levels had little or no effect. However, after depletion of adrenal tocopherol, even physiological concentrations of AA exerted prooxidant effects, stimulating LP. To assess the antioxidant potency of AA, its effects to inhibit ferrous ion (Fe2+)-induced LP were determined. Mitochondria from the outer zone contained far more alpha-tocopherol than those from the inner zone and were more sensitive to the antioxidant effects of AA. After tocopherol depletion, the antioxidant potency of AA in outer zone mitochondria decreased, but there was little change in the inner zone. The results indicate that the actions of AA are determined in part by mitochondrial tocopherol content, and, as a result, vary in the different zones of the adrenal cortex.     

Immunocytochemical mapping of basic fibroblast growth factor in the developing and adult rat adrenal gland

Summary We studied the spatial and temporal pattern of basic fibroblast growth factor (bFGF) immunoreactivity in the rat adrenal gland during postnatal development. In the cortex the glomerulosa zone reveals a strong anti-bFGF immunoreactivity at all developmental ages studied. In the fasciculata zone the high number of anti-bFGF immunoreactive cells in the first week decreases during the second and third week. The late developing reticularis zone shows only few anti-bFGF labeled cells at all postnatal ages. This distributional pattern of bFGF immunoreactivity matches that of mitotic activity in the rat adrenal cortex strengthening the role of bFGF as an autocrine growth factor for adrenocortical cells. In the medulla anti-bFGF positive chromaffin cells become detectable at postnatal day (P) 8 and increase in number during the second and third week. In the adult rat the staining intensity of the chromaffin cells was higher than at P18. In the adult medulla bFGF colocalizes with noradrenaline suggesting its presence in a chromaffin cell subpopulation. In accordance with previous results the role of the chromaffin cell bFGF as a neurotrophic factor for preganglionic sympathetic neurons is discussed.     

Adrenal cortex -- the next biological clock?

It is well known that plasma levels of dehydroepiandrosterone (DHEA), a steroid hormone secreted by zona reticularis (ZR) of the adrenal cortex, reach the maximal values in the third decade of life and then gradually decline with age. Moreover, the DHEA deficiency is probably responsible for several functional disturbances connected with aging. It was also found that ZR reaches its definitive volume at puberty and undergoes selective atrophy during the aging. Thus, the decline of DHEA may be a simple consequence of ZR atrophy in aged subjects. A hypothesis presented here attempts to explain the mechanism of the age-related ZR atrophy and is based on the adrenal cortex cell kinetics. In the adrenal cortex the cell proliferation indices are lower when we pass from zona glomerulosa (ZG) to the inner zones and are the lowest in ZR. In contrast, the apoptotic index is the highest in ZR. It is suggested that adrenocortical cells renew from the progenitor cells located in ZG /zona fasculata boundary and /or in subcapsular layer. These cells migrate centripetally undergoing the subsequent steps of differention and consecutive divisions - and - if not die en route - reach the most central localization in ZR. In consequence, ZR includes the "oldest" adrenocortical cells which probably in majority reached the "Hayflick's number" and cannot divide. This results in the preponderance of apoptosis over proliferation leading to progressive ZR atrophy followed by a decline of secretion of ZR-derived steroid hormones.     

Low-density lipoprotein receptor activity in the guinea pig adrenal cortex. II. Zonal response to aminoglutethimide and 17 alpha-ethinylestradiol

Low-density lipoprotein (LDL) receptor activity and the concentration of cholesterol were measured in the outer (glomerulosa/fasciculata) and inner (reticularis) zones of the adrenal cortex of the guinea pig to examine the relation between cholesterol content and LDL receptor activity. While the concentration of cholesterol was 2-3-times higher in the outer cortical zone, the maximum high-affinity binding capacity for LDL was essentially the same for the two zones, or slightly higher for the inner zone. Adrenocorticotrophic hormone (ACTH) caused a significant increase in LDL receptor activity only in the outer zone, but led to a reduction in the cholesterol content in both adrenocortical zones. The treatment of animals with 17 alpha-ethinyl-estradiol also resulted in a reduction of cholesterol in both adrenocortical zones, but an increase in LDL receptor number only in the outer zone. The latter effect was partially reversed by the administration of dexamethasone. Aminoglutethimide, which was used in a dose that did not block steroidogenesis but did block the hydrolysis of cholesteryl esters in response to ACTH, did not prevent the ACTH-induced increase in LDL receptor number in the outer zone. Thus, the number of LDL receptors was increased in the zona fasciculata by ACTH in the absence of a reduction in cellular cholesterol content, while the number of LDL receptors in the zona reticularis was not increased by ACTH even in the face of a reduction in cellular cholesterol. Exclusive of the experiments employing aminoglutethimide, when the cellular cholesterol content was plotted against LDL binding activity, an excellent inverse correlation was revealed for the zona fasciculata, but essentially no correlation was noted for the zona reticularis. It is concluded that the outer and inner cortical zones of the guinea pig adrenal are quite distinct in the nature of their LDL receptor activity and regulation: the LDL receptor of the outer zone appears to function in a way similar to what has been reported for the whole adrenal cortex of other species in that receptor number correlates with tissue cholesterol content and is primarily regulated by ACTH; the LDL receptor number of the inner zone, however, does not correlate with tissue cholesterol content and is apparently not regulated by ACTH.     

Immunohistochemical localization of adrenal ferredoxin in bovine adrenal cortex.

Adrenal ferredoxin, the iron-sulfur protein associated with cytochrome P-450 in adrenocortical mitochondria, has been localized at the light microscopic level in bovine adrenal cortex. Localization was achieved through the use of rabbit antiserum to bovine adrenal ferrodoxin in an unlabeled antibody peroxidase-antiperoxidase method. When sections of bovine adrenal glands were exposed to the adrenal ferredoxin antiserum, intense staining was observed in parenchymal cells of the three cortical zones. Staining for adrenal ferredoxin was not detected in the medullary chromaffin cells. The presence of adrenal ferredoxin in the three cortical zones was verified by electron paramagnetic resonance spectrometry. These determinations also revealed that while the zona fasciculata and the zona reticularis contained approximately equal concentrations of adrenal ferredoxin, the concentration of the iron-sulfur protein in the zona glomerulosa was considerably lower. Similar results were obtained when the levels of cytochrome P-450 were determined in the three cortical zones. These results represent the first immunohistochemical localization within an intact tissue or cell of any component of an NADPH-dependent electron transport sequence which is responsible for the reduction of cytochrome P-450.     

Spontaneous or experimentally induced formation of a special zone in the adrenal cortex of the adult brush-tailed possum (Trichosurus vulpecula)

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 follicle-stimulating 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 (closest 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 induced after 14 days of FSH treatment given to 2-month castrated males. In all castrated and FSH-treated 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.     

An inverse correlation between expression of a preprocathepsin B-related protein with cysteine-rich sequences and steroid 11beta -hydroxylase in adrenocortical cells

A cDNA encoding a secretory protein hitherto unknown was cloned from mouse adrenocortical cells by subtractive hybridization between the cells without and with expressing steroid 11beta-hydroxylase (Cyp11b-1), a marker for the functional differentiation of cells in the zonae fasciculata reticularis (zFR). The deduced protein consisting of 466 amino acids contained a secretory signal, epidermal growth factor-like repeats, and a proteolytically inactive cathepsin B-related sequence. The amino acid sequence was 89% identical with that of human tubulointerstitial nephritis antigen-related protein. Among the mouse organs examined, adrenal glands prominently expressed its mRNA. The mRNA and its encoded protein were detected in the outer adrenocortical zones that do not express Cyp11b-1, i.e. the zona glomerulosa and the undifferentiated cell zone, while being undetectable in zFR that express Cyp11b-1. The new protein was designated as adrenocortical zonation factor 1 (AZ-1). Clonal lines with different levels of AZ-1 expression were established from Y-1 adrenocortical cells that originally express Cyp11b-1 but little AZ-1. Analyses of the clonal lines revealed that Cyp11b-1 is detected in the clonal lines maintaining little AZ-1 expression and becomes undetectable in those expressing AZ-1. On the other hand, irrespective of the AZ-1 expression, all clones expressed cholesterol side-chain cleavage enzyme, which occurs throughout the cortical zones. These results demonstrated that adrenocortical cells expressing AZ-1 do not express Cyp11b-1, whereas those with little AZ-1 express this zFR marker in vitro and in vivo, implying a putative role of AZ-1 in determining the zonal differentiation of adrenocortical cells.