Sex differences in adrenocortical structure and function

Summary Adrenal glands of adult female rats are heavier than the glands of corresponding male rats. Postpubertal orchiectomy increases the adrenal weight, an effect restored by testosterone replacement. Under the same conditions ovariectomy of 8 weeks duration does not change the adrenal weight while estradiol replacement enhances the relative adrenal weight.Karyometric studies have shown that nuclei in the female zona fasciculata cells are larger (app. 18%) than those of the male. Similar but only slight differences (2%) were observed in the zona reticularis. Orchiectomy results in enlargement of cell nuclei within all zones of the adrenal cortex; testosterone replacement has the opposite effect. Ovariectomy of 8 weeks duration slightly enhances the volume of nuclei of the zona glomerulosa cells, has no effect on the nuclei of the zona fasciculata and reduces the volume of nuclei in the zona reticularis. Estradiol replacement reduces the volume of nuclei of the zona glomerulosa cells but increases nuclear volume in the zona fasciculata and in the zona reticularis.Thus testosterone has an inhibitory effect on the adrenal cortex of the rat while the physiologic effect of estradiol on the morphology of this gland, particularly on the zona fasciculata cells is rather dubious.The author wishes to thank Mrs. B. Westerska and Miss K. Siejak for excellent technical assistance.This research was supported in part by a grant from the Zoological Committee, 2nd Department, Polish Academy of Sciences.This paper is dedicated to the memory of late Kazimierz Mietkiewski, M.D., Ph.D. whose encouragements and suggestions were most stimulating for my work.     

Sex differences in adrenocortical structure and function. VI. Long-term effect of gonadectomy and testosterone or estradiol replacement on rat adrenal cortex

Gonadectomy or sham operations were performed on rats of Wistar strain weighing 150--170 g. Animals were sacrificed in groups, males 397 days and females 395 days after surgery. 14 days before autopsy some of the gonadectomized rats were injected with a single dose of depo-testosterone or depo-estradiol respectively. Absolute as well as relative adrenal weights were larger in female than in male rats. Long-term orchiectomy did not change these weights while testosterone, if compared with control, lowers the weights of the adrenals. Long-term ovariectomy lowers the weights of the glands and estradiol reversed them to the control level. Adrenal cortex of rats studied show irregular zonation. Within the zona fasciculata two types of cells were observed--with strongly eosinophilic and pale cytoplasm. In some cases groups of hypertrophied cells surrounded by spindle-shaped cells were observed. Neither long-term gonadectomy nor gonadal hormone replacement has a marked effect on the structure of the adrenal cortex. In male and female rats there was no difference in the volume of single zona fasciculata and reticularis cells. Long-term orchiectomy has no effect on this volume while testosterone resulted in a lowering of volume of zona fasiculata cells. Neither long-term ovariectomy nor estradiol has an effect on morphometric parameters studied in the fasciculata and reticularis zones. Distinct sex differences and dependence on sex hormones were found in reactions for alpha-glycerophosphate dehydrogenase and nonspecific esterases activity.     

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.     

Immunocytochemical localization of adrenodoxin in bovine adrenal cortex by protein A-gold technique

To investigate the intracellular localization of the enzymes that are involved in steroid hormone synthesis, an immunocytochemical study of the distribution of adrenodoxin in cells of the bovine adrenal cortex was carried out by the post-embedding immunostaining method and by immuno-cryoultramicrotomy in combination with the protein A-gold technique. Gold particles were seen on the matrix and the inner membrane of all the mitochondria examined, which have typical vesicular or tubulo-vesicular cristae, in parenchymal cells of the zona fasciculata and the zona reticularis. Gold particles were distributed homogeneously among the mitochondria. The density of gold particles on mitochondria in the parenchymal cells of the zona glomerulosa was lower than that of the zona fasciculata, which was similar to that of the zona reticularis. Gold particles were also seen on round, electron-dense intramitochondrial bodies in the parenchymal cells. The intramitochondrial bodies were abundant in the zona glomerulosa and the outer region of the zona fasciculata.     

Sex differences in adrenocortical structure and function

Summary Adrenal glands of adult male hamsters are larger and secrete more cortisol than those of females. Stereology was therefore used to study zonal and cellular aspects of development of the adrenal cortex of male and female hamsters. Adrenal glands were studied at weekly intervals from day 21 to day 77 of postnatal ontogenesis. Within this period, body weight did not differ significantly between the sexes. During development, absolute and relative adrenal weights were higher in males; their zona glomerulosa (ZG), zona fasciculata (ZF) and zona reticularis (ZR) become markedly larger than those in females. No marked changes in the volume of individual ZG cells occurred although ZF cells and ZR cells become larger in male than female animals. The total number of adrenocortical cells increased within the period studied, a greater increase being observed in ZG and ZF in males. No distinct sex difference was observed in the number of ZR cells throughout development. From day 56 of postnatal life the adrenal cortex of male hamster contained more parenchymal cells than the female gland. These results thus indicate that sex differences in hamster adrenal cortex depend upon changes in number and size of parenchymal cells.Supported in part by a grant from the Committee of Zoology, Polish Academy of Sciences     

Histochemical localization of monoamine oxidase types A and B in the adrenal gland

Summary The distribution of monoamine oxidase types A and B within the adrenal galdn was studied in several mammals by histochemical methods. Controls showed that the methods were valid. The bovine adrenal medulla contained mostly the B type enzyme, distributed heterogeneously, with some A type associated with endothelium, nerves, and cells surrounding the nerves. The bovine adrenal cortex showed a marked zonation of the two types of monoamine oxidase. The zona glomerulosa contained the B type enzyme and the zona fasciculata and zona reticularis contained the type A enzyme. The adrenal medulla of the dog, cat, and rat demonstrated relatively little enzyme activity and it appeared to be both type A and B. The adrenal cortex of these animals appeared to contain mostly the B type enzyme, except the canine zona reticularis, which contained some A type monoamine oxidase as well.     

Immunocytochemical localization of adrenodoxin in bovine adrenal cortex by protein A-gold technique

Summary To investigate the intracellular localization of the enzymes that are involved in steroid hormone synthesis, an immunocytochemical study of the distribution of adrenodoxin in cells of the bovine adrenal cortex was carried out by the post-embedding immunostaining method and by immuno-cryoultramicrotomy in combination with the protein A-gold technique. Gold particles were seen on the matrix and the inner membrane of all the mitochondria examined, which have typical vesicular or tubulo-vesicular cristae, in parenchymal cells of the zona fasciculata and the zona reticularis. Gold particles were distributed homogeneously among the mitochondria. The density of gold particles on mitochondria in the parenchymal cells of the zona glomerulosa was lower than that of the zona fasciculata, which was similar to that of the zona reticularis. Gold particles were also seen on round, electron-dense intramitochondrial bodies in the parenchymal cells. The intramitochondrial bodies were abundant in the zona glomerulosa and the outer region of the zona fasciculata.Supported by a Grant-in-Aid for Scientific Research on Priority Areas (no. 63635505) from the Ministry of Education, Science and Culture of Japan     

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.     

Sex differences in adrenocortical structure and function. V. The effects of postpubertal gonadectomy and gonadal hormone replacement on nuclear-cytoplasmic ratio, morphology and histochemistry of rat adrenal cortex.

Studies were carried out on adult rats of Wistar strain. Six weeks after postpubertal gonadectomy some of the orchiectomized rats were injected with a single dose of testosterone cypionate and ovari-ctomized rats with estradiol cypionate (17 beta-cyclopentyloproprionate esters of testosterone or estradiol). The control rats were sham operated. Investigations were carried out 8 weeks after surgery. Absolute and relative adrenal weight is lower in the male than in the female rat. Orchiectomy increases these weights while testosterone restores them to their normal level. Ovariectomy has no effect on the weight of adrenal, estradiol, however, increases the relative weight of the gland. The adrenal cortex of the adult female is wider than in the adult male rat and in female gland sudanophobic zone is lacking. Orchiectomy leads to the broadening of the adrenal cortex and testosterone replacement has an opposite effect. Ovariectomy has no effect on the structure of adrenal cortex and estradiol replacement resulted in the narrowing of zona glomerulosa. There were no differences in the nuclear-cytoplasmic ratio of zona glomerulosa cells in male and Female rats and neither gonadectomy nor gonadal hormone replacement has no effect on this parameter. The nuclear-cytoplasmic ratio of zona fasciculata cells in the male is markedly higher than in female. Orchiectomy lowers this ratio and testosterone restores it to the normal level. Neither ovariectomy nor estradiol replacement has effect on the nuclear-cytoplasmic ratio of zona fasciculata cells. Similar changes as those in the zona fasciculata were observed in zona reticularis cells. Among the oxidoreductases studied, the most marked sex differences were found in alpha-glycerophosphate dehydrogenase activity. In control female rats an intense reaction for this enzyme is observed in broad band of cells of the zona fasciculata interna, while in male rats only individual cells showed this reaction. In orchiectomized rats the reaction for this enzyme is similar as in control female rats and testosterone restores it to normal. Ovariectomy has no effect on localisation of reaction while after estradiol replacement reaction was more intense. Regarding the remaining oxidoreductases studied, in the adrenal cortex of rats of both sexes no marked differences were observed in localization, however, intensity of reaction depended upon applied experimental conditions. More distinct sex differences were observed in topochemistry of some hydrolases and intensity of reaction depended upon the applied experimental conditions.     

Proliferation and distribution of adrenocortical cells in ACTH treated female hamsters

The aim of the study was the analysis, by means of combined stereologic methods and metaphase arrest technique of the adrenocortical growth of ACTH treated hamster. Adult female hamsters were treated daily with 50 micrograms ACTH (Synacthen Depot, Ciba) for 3, 6 and 9 days. Between days 3 and 9 nearly linear increase in adrenal gland weight was observed, mainly due to the enlargement of the fasciculata and reticularis zones. Throughout the experiment, there was no marked change in the number of the zona glomerulosa cells while from the day 3 a notable increase in the number of fasciculata and reticularis cells occurred. Prolonged ACTH treatment resulted in a significant increase in 3H-thymidine incorporation with the highest value on day 6 and subsequent drop on day 9. Also metaphase index of adrenocortical cells notably increased due to ACTH administration and again the highest value was found on day 6 of experiment. From stereologic data and from metaphase index, adrenocortical cell birth-rates in individual adrenocortical zones and in the entire cortex were calculated. There were great differences between the values calculated from metaphase index and those obtained from stereologic data, with the former significantly lower. ACTH enhances centripetal migration of parenchymal cells in the hamster adrenal cortex and causes their accumulation in the zona reticularis.     

Steroidogenic characteristics of the adrenal cortex of the mare studied by electron microscopy

The three concentric zones of the horse adrenal cortex (zonae glomerulosa, fasciculata and reticularis) showed marked interpenetration and exhibited a different relative development according to their position in the gland. Whereas the three cortical zones each had a specific histological structure, the ultrastructure of their cells showed a certain qualitative homogeneity. The differences corresponded essentially to the relative abundance of the constituents which are generally considered typical of steroidogeneous cells: mitochondria with vesicular cristae, smooth endoplasmic reticulum and lipid droplets. Their importance increased progressively from the zona glomerulosa to the zona reticularis. In this zone, the presence of gap and septate-like cell junctions, and mitochondria with vesicular cristae in close proximity to a smooth endoplasmic reticulum with numerous dilated tubules, suggested that steroidogenesis may be the most active. Ultrastructural findings were indicative of only quantitative differences between the steroidogenic capacities of the three zones of the mare adrenal cortex.     

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.     

Stimulation of the adrenal gland of the mongolian gerbil after ovariectomy

The effect of ovariectomy on the adrenal gland was studied in the Mongolian gerbil. Castration stimulated cells in the zona fasciculata as well as those in the region between the zona fasciculata and zona reticularis. No alterations occurred in the zona reticularis. The width of the intermediate region was enlarged, the cells were hypertrophic and contained an increased number of concentric whorls of rough endoplasmic reticulum. Smooth endoplasmic reticulum was hypertropic in cells of the zona fasciculata and intermediate region. Many mitochondria in cells of the intermediate region were larger than those in controls. Stimulation of cellular ultrastructure is directly correlated with a significant elevation in plasma cortisol in ovariectomized gerbils.     

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.     

The influence of castration on cell proliferation in the adrenal cortex of female rats

Proliferation kinetics in the zones of the adrenal cortex of female rats after castration are reported. Following ovariectomy a wave of increased cell proliferation was observed in the zona glomerulosa and zona fasciculata after 1 and 2 days respectively. Increased cell proliferation in the zona reticularis was only slight. Cell proliferation returned to the level in control animals at 4 weeks. It is assumed that the regulative process following ovariectomy takes place directly in the adrenal cortex but it is possible that steroid metabolism in the liver is of importance. No relationship between the 3H-TdR labelling index (LI) and the mitotic index (MI) was observed.     

Effects of lactation on the border zone formation in the female mastomys (Praomys coucha) adrenal cortex.

Adrenocortical zonation and especially border zone formation were investigated in relation to reproduction in female mastomys of the wild-colored inbred strain (MWC). The adrenal weight and the whole thickness of the cortex remained relatively constant throughout pregnancy, delivery and lactation. During pregnancy the zona fasciculata increased, but the z. reticularis decreased in thickness. The border zone between the z. fasciculata and the z. reticularis thickened 2-fold during pregnancy and further 2-fold during lactation. This zone was composed of vacuolated cell layers and nodules in females with a normal reproductive career. In young virgins this zone was barely identified by the presence of a few highly vacuolated cells. These cells became prominent during pregnancy and lactation. During lactation two types of cell aggregations, distinguishable by their stainability, vacuolation, localization and cellularity, appeared in the thickened border zone. The nodules first appeared within the z. reticularis after lactation. Neither thickening of both border zone and z. fasciculata nor formation of nodules occurred when lactation was interrupted after delivery. Thus, the changes in the female MWC adrenal, above all the appearance of nodules, seemed to be more dependent upon lactation than pregnancy. The vacuolated cells and the nodules might originate from the thickened z. fasciculata and the thinned z. reticularis, respectively, by the specific metamorphosis of the cells adjacent to the border zone. Their fusion and induction of fibrous tissue migration may result in the wider border zone.     

Effect of biseptol on the adrenal cortex of the white rat in postoperative shock

The study aimed to find out the effect of sulfonamide combined with Trimetaprim-Biseptol 480 on the adrenal cortex in post-operative shock after removal of SPIGELian lobe (lobectomy of the lobus caudatus and unilaterally of one kidney with its suprarenal gland. The study was performed on a material of white rats which were post-operatively administered Biseptol 480 in doses 5 times bigger than those given to men. It was attempted to determine histochemically the intensity of the adrenal cortex' function by testing the number of lipid droplets, activity of the main enzyme of steroidogenesis (beta-hydroxy steroid dehydrogenase) and the level of alpha-ketols (as the final stage of steroidogenesis). Pathomorphologic examinations were also performe. On the basis of the present study's results, it was observed that - in the case of liver-lobectomy - the zona fasciculata and zona reticularis are functionally stimulated but the zona glomerulosa becomes insufficient. In the case of nephrectomy plus suprarenal gland's removal, all the adrenal cortex becomes insufficient. Administration of Biseptol in the 1st case contributed to hormonal inactivation of the zone glomerulosa cells, but in the 2nd case, it caused an increased activity of steroid dehydrogenase and an increase of the alpha-ketol level in the zona fasciculata.