Aging induces region-specific dysregulation of hormone synthesis in the primate adrenal gland.

Adrenal glands, vital for steroid secretion and the regulation of metabolism, stress responses and immune activation, experience age-related decline, impacting systemic health. However, the regulatory mechanisms underlying adrenal aging remain largely uninvestigated. Here we established a single-nucleus transcriptomic atlas of both young and aged primate suprarenal glands, identifying lipid metabolism and steroidogenic pathways as core processes impacted by aging. We found dysregulation in centripetal adrenocortical differentiation in aged adrenal tissues and cells in the zona reticularis region, responsible for producing dehydroepiandrosterone sulfate (DHEA-S), were highly susceptible to aging, reflected by senescence, exhaustion and disturbed hormone production. Remarkably, LDLR was downregulated in all cell types of the outer cortex, and its targeted inactivation in human adrenal cells compromised cholesterol uptake and secretion of dehydroepiandrosterone sulfate, as observed in aged primate adrenal glands. Our study provides crucial insights into endocrine physiology, holding therapeutic promise for addressing aging-related adrenal insufficiency and delaying systemic aging.

Female baboon adrenal zona fasciculata and zona reticularis regulatory and functional proteins decrease across the life course.

Debate exists on life-course adrenocortical zonal function trajectories. Rapid, phasic blood steroid concentration changes, such as circadian rhythms and acute stress responses, complicate quantification. To avoid pitfalls and account for life-stage changes in adrenocortical activity indices, we quantified zonae fasciculata (ZF) and reticularis (ZR) across the life-course, by immunohistochemistry of key regulatory and functional proteins. In 28 female baboon adrenals (7.5-22.1 years), we quantified 12 key proteins involved in cell metabolism, division, proliferation, steroidogenesis (including steroid acute regulatory protein, StAR), oxidative stress, and glucocorticoid and mitochondrial function. Life-course abundance of ten ZF proteins decreased with age. Cell cycle inhibitor and oxidative stress markers increased. Seven of the 12 proteins changed in the same direction for ZR and ZF. Importantly, ZF StAR decreased, while ZR StAR was unchanged. Findings indicate ZF function decreased, and less markedly ZR function, with age. Causes and aging consequences of these changes remain to be determined.

Accelerated telomere shortening in adrenal zona reticularis in patients with prolonged critical illness.

Background: The number of patients with prolonged critical illness (PCI) has been increasing in many countries, and the adrenal gland plays an important role in maintaining homeostasis during PCI. Chronic disease burden is reportedly associated with shorter telomere lengths in human tissues. Telomere shortening in human somatic cells is largely dependent on cell divisions, and critically short telomeres lead to cellular dysfunction and aging. However, the association between PCI and telomere lengths in human adrenal cells is poorly understood. In this study, we investigated this association to assess whether the burden of PCI could accelerate the aging process in adrenal cells. Methods: Adrenocortical tissues from patients who died after PCI usually show a diffuse pattern of intracellular cholesterol ester depletion (i.e., lipid depletion). This study examined near-normal adrenal glands obtained from autopsied patients who died suddenly (control group) and lipid-depleted adrenal glands obtained from autopsied patients who died after PCI (PCI group). The control group included 7 men aged 80 to 94 years (mean age: 85.3 years) and 7 women aged 84 to 94 years (mean age: 87.7 years). The PCI group included 10 men aged 71 to 88 years (mean age: 78.8 years) and 8 women aged 77 to 95 years (mean age: 85.6 years). By using quantitative fluorescence in situ hybridization, relative telomere lengths (RTLs) were determined in the parenchymal cells of the three adrenocortical zones (zona glomerulosa, zona fasciculata, and zona reticularis [ZR]) and in the chromaffin cells of the medulla. The number of adrenal parenchymal cells was determined by immunohistochemistry and digital image analysis. Results: RTLs in ZR cells were significantly shorter in the PCI group than in the control group for both men and women (P = 0.0001 for men and P = 0.0012 for women). However, RTLs in the remaining three types of adrenal cells did not differ between the control and PCI groups for both men and women. The number of ZR cells was higher in the PCI group than in the control group for both men and women (P < 0.0001 for both men and women). The proportion of the number of ZR cells to the total number of adrenocortical parenchymal cells was also higher in the PCI group than in the control group (P < 0.0001 for both men and women). The Ki-67 proliferation index in ZR cells was higher in the PCI group than in the control group (P = 0.0039 for men and P = 0.0063 for women). Conclusions: This study demonstrated ZR cell-specific telomere shortening in patients with adrenal lipid depletion who died after PCI. Our results suggest that the reactive proliferation of ZR cells accelerates the telomere shortening and aging process in ZR cells in these patients. The results of our study may contribute to the understanding of adrenal aging during PCI.

Cholesterol Deprivation Drives DHEA Biosynthesis in Human Adrenals.

Adrenarche is an early event in sexual maturation in prepubertal children and corresponds to the postnatal development of the adrenocortical zona reticularis (zR). However, the molecular mechanisms that govern the onset and maturation of zR remain unknown. Using tissue laser microdissection combined with transcript quantification and immunodetection, we showed that the human zR receives low levels of cholesterol in comparison with other adrenal layers. To model this metabolic condition, we challenged adrenal cells in vitro using cholesterol deprivation. This resulted in reprogramming the steroidogenic pathway toward inactivation of 3-beta-hydroxysteroid dehydrogenase type 2 (HSD3B2), increased CYB5A expression, and increased biosynthesis of dehydroepiandrosterone (DHEA), 3 key features of zR maturation during adrenarche. Finally, we found that cholesterol deprivation leads to decreased transcriptional activity of POU3F2, which normally stimulates the expression of HSD3B2 by directly binding to its promoter. These findings demonstrate that cholesterol deprivation can account, at least in part, for the acquisition of a zR-like androgenic program in humans.

Development of Human Adrenocortical Adenoma (HAA1) Cell Line from Zona Reticularis.

The human adrenal cortex is composed of distinct zones that are the main source of steroid hormone production. The mechanism of adrenocortical cell differentiation into several functionally organized populations with distinctive identities remains poorly understood. Human adrenal disease has been difficult to study, in part due to the absence of cultured cell lines that faithfully represent adrenal cell precursors in the early stages of transformation. Here, Human Adrenocortical Adenoma (HAA1) cell line derived from a patient's macronodular adrenocortical hyperplasia and was treated with histone deacetylase inhibitors (HDACis) and gene expression was examined. We describe a patient-derived HAA1 cell line derived from the zona reticularis, the innermost zone of the adrenal cortex. The HAA1 cell line is unique in its ability to exit a latent state and respond with steroidogenic gene expression upon treatment with histone deacetylase inhibitors. The gene expression pattern of differentiated HAA1 cells partially recreates the roster of genes in the adrenal layer that they have been derived from. Gene ontology analysis of whole genome RNA-seq corroborated increased expression of steroidogenic genes upon HDAC inhibition. Surprisingly, HDACi treatment induced broad activation of the Tumor Necrosis Factor (TNF) alpha pathway. This novel cell line we developed will hopefully be instrumental in understanding the molecular and biochemical mechanisms controlling adrenocortical differentiation and steroidogenesis.

Low-Dose Exposure to Endocrine Disruptor Dichlorodiphenyltrichloroethane (DDT) Affects Transcriptional Regulation of Adrenal Zona Reticularis in Male Rats.

The study examined transcriptional regulation of adrenal zona reticularis development in male Wistar rats exposed to low doses of endocrine disruptor dichlorodiphenyltrichloroethane (DDT) prenatally and postnatally. At pubertal age, zona reticularis demonstrated a retarded growth with a low proliferative activity of its cells. At this age, zona reticularis was characterized with overexpression of ß-catenin by steroid-producing cells; a high percentage of cells with membrane and cytoplasmic localization of ß-catenin, and reduced number of cells with nuclear ß-catenin attesting to insufficient activation of Wnt signaling. Expression of transcriptional factor Oct4 by reticularis cells was down-regulated indicating their diminished proliferative potency. No significant alterations in Sonic Hedgehog expression were observed. Thus, DDT-provoked disorders of transcriptional regulation result in abnormal development of zona reticularis thereby disturbing sexual maturation in males.

Adrenal androgens, adrenarche, and zona reticularis: A human affair?

In humans, reticularis cells of the adrenal cortex fuel the production of androgen steroids, constituting the driver of numerous morphological changes during childhood. These steps are considered a precocious stage of sexual maturation and are grouped under the term "adrenarche". This review describes the molecular and enzymatic characteristics of the zona reticularis, along with the possible signals and mechanisms that control its emergence and the associated clinical features. We investigate the differences between species and discuss new studies such as genetic lineage tracing and transcriptomic analysis, highlighting the rodent inner cortex's cellular and molecular heterogeneity. The recent development and characterization of mouse models deficient for Prkar1a presenting with adrenocortical reticularis-like features prompt us to review our vision of the mouse adrenal gland maturation. We expect these new insights will help increase our understanding of the adrenarche process and the pathologies associated with its deregulation.

The Cost of Adaptation to Information Loading in Mice with Various Genetic Profiles.

It was found that male BALB/c and F1(C57BL/6×DBA/2) mice are able to recognize the structure of a complex food-gathering task, when modeling the information loading similar to intellectual work in humans. There were significant differences between linear and hybrid animals in the pattern of learning process formation and prevailing psychoemotional reactions that accompany information load. Factors of information loading (uncertainty of maze environment and solution of the food-gathering task) had a specific influence on the CNS and manifested in individual non-specific features. The presented experimental conditions (changes in the metabolic and functional state) revealed pronounced intergroup differences in the reaction of the functional zones of the adrenal cortex. In hybrid mice, information loading induced a significant decrease in testosterone level and thickness of the zona reticularis producing precursor hormones. This is probably due to disruption of interactions in the adrenal-thyroid system in hybrid mice, whereas in BALB/c mice, these interactions fully protect from suppression of testosterone production, the main anabolic hormone. The individual characteristics of the response to information loading can be formed as a result of unequal involvement of the psychophysiological, psychological, and autonomic systems responsible for adaptation to environmental factors.

The Age-Dependent Changes of the Human Adrenal Cortical Zones Are Not Congruent.

BACKGROUND: While previous studies indicate that the zonae reticularis (ZR) and glomerulosa (ZG) diminish with aging, little is known about age-related transformations of the zona fasciculata (ZF). OBJECTIVES: To investigate the morphological and functional changes of the adrenal cortex across adulthood, with emphasis on (i) the understudied ZF and (ii) sexual dimorphisms. METHODS: We used immunohistochemistry to evaluate the expression of aldosterone synthase (CYP11B2), visinin-like protein 1 (VSNL1), 3ß-hydroxysteroid dehydrogenase type II (HSD3B2), 11ß-hydroxylase (CYP11B1), and cytochrome b5 type A (CYB5A) in adrenal glands from 60 adults (30 men), aged 18 to 86. Additionally, we employed mass spectrometry to quantify the morning serum concentrations of cortisol, 11-deoxycortisol (11dF), 17α-hydroxyprogesterone, 11-deoxycorticosterone, corticosterone, and androstenedione in 149 pairs of age- and body mass index-matched men and women, age 21 to 95 years. RESULTS: The total cortical area was positively correlated with age (r = 0.34, P = 0.008). Both the total (VSNL1-positive) and functional ZG (CYP11B2-positive) areas declined with aging in men (r = -0.57 and -0.67, P < 0.01), but not in women. The CYB5A-positive area declined with age in both sexes (r = -0.76, P < 0.0001). In contrast, the estimated ZF area correlated positively with age in men (r = 0.59, P = 0.0006) and women (r = 0.49, P = 0.007), while CYP11B1-positive area remained unchanged across ages. Serum cortisol, corticosterone, and 11-deoxycorticosterone levels were stable across ages, while 11dF levels increased slightly with age (r = 0.16, P = 0.007). CONCLUSION: Unlike the ZG and ZR, the ZF and the total adrenal cortex areas enlarge with aging. An abrupt decline of the ZG occurs with age in men only, possibly contributing to sexual dimorphism in cardiovascular risk.

Adrenal cortex development and related disorders leading to adrenal insufficiency.

The adult human adrenal cortex produces steroid hormones that are crucial for life, supporting immune response, glucose homeostasis, salt balance and sexual maturation. It consists of three histologically distinct and functionally specialized zones. The fetal adrenal forms from mesodermal material and produces predominantly adrenal C19 steroids from its fetal zone, which involutes after birth. Transition to the adult cortex occurs immediately after birth for the formation of the zona glomerulosa and fasciculata for aldosterone and cortisol production and continues through infancy until the zona reticularis for adrenal androgen production is formed with adrenarche. The development of this indispensable organ is complex and not fully understood. This article gives an overview of recent knowledge gained of adrenal biology from two perspectives: one, from basic science studying adrenal development, zonation and homeostasis; and two, from adrenal disorders identified in persons manifesting with various isolated or syndromic forms of primary adrenal insufficiency.

Changes in Adrenal Androgens and Steroidogenic Enzyme Activities From Ages 2, 4, to 6 Years: A Prospective Cohort Study.

CONTEXT: The levels of adrenal androgens are increased through the action of steroidogenic enzymes with morphological changes in the adrenal zona reticularis. OBJECTIVE: We investigated longitudinal changes in androgen levels and steroidogenic enzyme activities during early childhood. DESIGN AND PARTICIPANTS: From a prospective children's cohort, the Environment and Development of Children cohort, 114 boys and 86 girls with available blood samples from ages 2, 4, and 6 years were included. OUTCOME MEASUREMENTS: Serum concentrations of adrenal androgens using liquid chromatography-tandem mass spectrometry and steroidogenic enzyme activity calculated by the precursor/product ratio. RESULTS: During ages 2 to 4 years, 17,20-lyase and dehydroepiandrosterone (DHEA) sulfotransferase activities increased (P < 0.01 for both in boys). During ages 4 to 6 years, 17,20-lyase activity persistently increased, but 3ß-hydroxysteroid dehydrogenase (HSD) and 17ß-HSD activities decreased (P < 0.01 for all). Serum DHEA sulfate (DHEA-S) levels persistently increased from 2, 4, to 6 years, and DHEA, 17-hydroxyprogesterone, and androstenedione levels increased during ages 4 to 6 years (P < 0.01 for all). Serum DHEA-S levels during early childhood were associated with body mass index z-scores (P = 0.001 in only boys). CONCLUSION: This study supports in vivo human evidence of increased 17,20-lyase and DHEA sulfotransferase activities and decreased 3ß-HSD activity during early childhood.

Update on adrenarche.

PURPOSE OF REVIEW: Adrenarche is the pubertal maturation of the innermost zone of the adrenal cortex, the zona reticularis. The onset of adrenarche occurs between 6 and 8 years of age when dehydroepiandrosterone sulfate (DHEAS) concentrations increase. This review provides an update on adrenal steroidogenesis and the differential diagnosis of premature development of pubic hair. RECENT FINDINGS: The complexity of adrenal steroidogenesis has increased with recognition of the alternative 'backdoor pathway' and the 11-oxo-androgens pathways. Traditionally, sulfated steroids such as DHEAS have been considered to be inactive metabolites. Recent data suggest that intracellular sulfated steroids may function as tissue-specific intracrine hormones particularly in the tissues expressing steroid sulfatases such as ovaries, testes, and placenta. SUMMARY: The physiologic mechanisms governing the onset of adrenarche remain unclear. To date, no validated regulatory feedback mechanism has been identified for adrenal C19 steroid secretion. Available data indicate that for most children, premature adrenarche is a benign variation of development and a diagnosis of exclusion. Patients with premature adrenarche tend to have higher BMI values. Yet, despite greater knowledge about C19 steroids and zona reticularis function, much remains to be learned about adrenarche.

DHEAS and Human Development: An Evolutionary Perspective.

Adrenarche, the post-natal rise of DHEA and DHEAS, is unique to humans and the African Apes. Recent findings have linked DHEA in humans to the development of the left dorsolateral prefrontal cortex (LDPFC) between the ages of 4-8 years and the right temporoparietal junction (rTPJ) from 7 to 12 years of age. Given the association of the LDLPFC with the 5-to-8 transition and the rTPJ with mentalizing during middle childhood DHEA may have played an important role in the evolution of the human brain. I argue that increasing protein in the diet over the course of human evolution not only increased levels of DHEAS, but linked meat consumption with brain development during the important 5- to-8 transition. Consumption of animal protein has been associated with IGF-1, implicated in the development of the adrenal zona reticularis (ZR), the site of DHEAS production. In humans and chimps, the zona reticularis emerges at 3-4 years, along with the onset of DHEA/S production. For chimps this coincides with weaning and peak synaptogenesis. Among humans, weaning is completed around 2 ½ years, while synaptogenesis peaks around 5 years. Thus, in chimpanzees, early cortical maturation is tied to the mother; in humans it may be associated with post-weaning provisioning by others. I call for further research on adrenarche among the African apes as a critical comparison to humans. I also suggest research in subsistence populations to establish the role of nutrition and energetics in the timing of adrenarche and the onset of middle childhood.

The Adrenal Cortex, an Underestimated Site of SARS-CoV-2 Infection.

Background: The majority of the critically ill patients may have critical illness-related corticosteroid insufficiency (CIRCI). The therapeutic effect of dexamethasone may be related to its ability to improve cortical function. Recent study showed that dexamethasone can reduce COVID-19 deaths by up to one third in critically ill patients. The aim of this article is to investigate whether SARS-CoV-2 can attack the adrenal cortex to aggravate the relative adrenal insufficiency. Methods: We summarized the clinical features of COVID-19 reported in currently available observational studies. ACE2 and TMPRSS2 expression was examined in human adrenal glands by immunohistochemical staining. We retrospectively analyzed serum cortisol levels in critically ill patients with or without COVID-19. Results: High percentage of critically ill patients with SARS-COV-2 infection in the study were treated with vasopressors. ACE2 receptor and TMPRSS2 serine protease were colocalized in adrenocortical cells in zona fasciculata and zona reticularis. We collected plasma cortisol concentrations in nine critically ill patients with COVID-19. The cortisol levels of critically ill patients with COVID-19 were lower than those in non-COVID-19 critically ill group. Six of the nine COVID-19 critically ill patients had random plasma cortisol concentrations below 10 µg/dl, which met the criteria for the diagnosis of CIRCI. Conclusion: We demonstrate that ACE2 and TMPRSS2 are colocalized in adrenocortical cells, and that the cortisol levels are lower in critically ill patients with COVID-19 as compared to those of non-COVID-19 critically ill patients. Based on our findings, we recommend measuring plasma cortisol level to guide hormonal therapy.

Regulation of Proliferative Processes in Rat Adrenal Cortex by Transcriptional Factor PRH under Conditions of Developmental Exposure to Endocrine Disruptor DDT.

The effects of endocrine disrupters of transcriptional control of morphogenesis are poorly studied. Changes in the expression of transcriptional factor PRH and proliferation of adrenal cortical cells were analyzed in pubertal and postpubertal rats exposed prenatally and postnatally to low doses of endocrine disrupter DDT. In rats exposed to DDT, the expression of PRH and proliferation of adrenal cortical cells differed from those in control rats. Association between these parameters was weakened in the zona glomerulosa and zona reticularis and was absent in the zona fasciculata. These findings suggest that exposure to DDT in pre- and postnatal periods impairs the regulation of proliferative processes by transcriptional factor PRH in all zones of rat adrenal cortex, which can be a mechanism of the disruptive action of DDT.

Biomolecular engineering of antidehydroepiandrosterone antibodies: a new perspective in cancer diagnosis and treatment using single-chain antibody variable fragment.

AIM: To develop a monoclonal antibody against dehydroepiandrosterone (DHEA) and miniaturize it, generating a single-chain antibody variable fragment (scFv) against DHEA as an adrenocortical carcinoma (ACC) marker. MATERIAL & METHODS: DHEA conjugated to keyhole limpet hemocyanin was used as an immunogen to obtain anti-DHEA hybridomas. Variable fragments were cloned from hybridoma 5B7 total RNA, and used to detect DHEA in normal adrenal tissue and ACC cells. RESULTS: IgM monoclonal antibody was highly specific, and the recombinant scFv preserved parental antibody characteristics, allowing tissue localization of DHEA. CONCLUSION: Undefined small lesions are challenges for clinicians and impact clinical adrenocortical tumor management. Generating an anti-DHEA scFv facilitates development of imaging tests for early diagnosis of pediatric ACC.

11-Ketotestosterone Is the Dominant Circulating Bioactive Androgen During Normal and Premature Adrenarche.

Context: Adrenarche refers to the rise of dehydroepiandrosterone sulfate (DHEA-S) associated with the development of a functional adrenal zona reticularis. Clinical features of adrenarche include onset of body odor, axillary hair, and pubic hair, which reflect increased androgen action. An early rise in adrenal androgens, or premature adrenarche (PremA), is a risk factor for adverse metabolic profiles in adolescence and adulthood. The bioactive androgens associated with adrenarche and PremA remain poorly understood. The adrenal gland is a potential source of testosterone (T) and the 11-oxygenated derivatives 11ß-hydroxytestosterone (11OHT) and 11-ketotestosterone (11KT). Objective: The objective of this study was to characterize the adrenal androgen biome contributing to adrenarche and PremA. Participants and Methods: With the use of mass spectrometry, 19 steroids including the 11-oxygenated derivatives of T were measured in sera obtained from girls with PremA (n = 37; 4 to 7 years) and age-matched girls (n = 83; 4 to 10 years). Results: In reference population girls, dehydroepiandrosterone, DHEA-S, androstenediol-3-sulfate, T, and 11KT all increased at the onset of adrenarche (6 to 8 years) and beyond (9 to 10 years) (P < 0.05 vs younger subjects 4 to 5 years). T, 11OHT, and 11KT were further elevated in PremA vs age-matched girls (P < 0.001). Circulating concentrations of 11KT during adrenarche and PremA exceeded those of T and 11OHT (11KT > T ≥ 11OHT). Androgen receptor activity and nuclear translocation studies demonstrated that 11KT is a potent androgen similar to T. Conclusions: Our findings suggest that 11KT is the dominant bioactive androgen in children during adrenarche and PremA. Its androgenic capacity suggests that it may be responsible for the phenotypic changes seen in these phenomena.

Human Adrenal Cortex: Epigenetics and Postnatal Functional Zonation.

The human adrenal cortex, involved in adaptive responses to stress, fluid homeostasis, and secondary sexual characteristics, arises from a tightly regulated development of a zone and cell type-specific secretory pattern. However, the molecular mechanisms governing adrenal zonation, particularly postnatal zona reticularis development, which produce adrenal androgens in a lifetime-specific manner, remain poorly understood. Epigenetic events, including DNA and histone modifications as well as regulation by noncoding RNAs, are crucial in establishing or maintaining the expression pattern of specific genes and thus contribute to the stability of a specific differentiation state. Emerging evidence points to epigenetics as another regulatory layer that could contribute to establishing the adrenal zone-specific pattern of enzyme expression. Here, we outline the developmental milestones of the human adrenal cortex, focusing on current advances and understanding of epigenetic regulation of postnatal functional zonation. Numerous questions remain to be addressed emphasizing the need for additional investigations to elucidate the role of epigenetics in the human adrenal gland. Ultimately, improved understanding of the epigenetic factors involved in adrenal development and function could lead to novel therapeutic interventions.

Stress-induced changes in the aged-rat adrenal cortex. Histological and histomorphometric study.

BACKGROUND: Stress exposure exerts direct effects on the morphology and functionality of the adrenal cortex. In addition, ageing effects growth, differentiation, apoptosis and cellularity of the cortex. The missing data is the combined effect of stress and ageing on the adrenal cortex. The aim of this study was to demonstrate the structural changes in the adrenal cortex following the exposure to stress in the adult and aged albino rats. MATERIALS AND METHODS: Forty rats were divided into groups I and II (adult and senile). Each group was further subdivided into subgroups a and b (control and stressed). Light and electron microscopic studies were done. Area per cent of collagen fibres (Masson's trichrome-stained sections), number of proliferating cells (optical density immunoreactivity in the Ki67 stained sections) and thickness of the three adrenal zones were also measured. RESULTS: Lamellar separation of the capsule with subcapsular spindle cell hyperplasia and areas of ghost cells were observed in zona glomerulosa (ZG) and zona fasciculata (ZF) in group I-b. Separation and indentation of the capsule with its lamellar separation were observed in group II-a with the existence of multiple scattered degenerative foci in ZF and zona reticularis (ZR). Similar and aggressive was the architectural pattern of ZF in group II-b with the presence of areas of homogenous degeneration. The nuclei of ZG had marginated chromatin in group I-b and were pyknotic with deformed irregular outlines in group II-b. Multiple lysosomes and vacuolar degeneration mitochondria were also seen in group I-b. The nuclei of ZF were irregular with condensed marginated heterochromatin in group I-b, irregular with scattered chromatin in group II-a and indented with areas of chromatin destruction in group II-b. Mitochondria with disrupted cristae and cristolysis were also detected in group I-b. Numerous lipofuscin granules and dilated smooth endoplasmic reticulum were revealed in group II-b. The mean collagen fibre area per cent and the mean number of the proliferating cells in group II-b were significantly higher by 39% and 23%. The thickness of ZG decreased significantly by 20% in group I-b. Contrary, the thickness of both ZF and ZR increased significantly by 10% in group I-b. CONCLUSIONS: Histological alterations occurred in the adrenal cortex in response to stress, especially when coupled with the advance of age. This was accompanied by increase in the area per cent of collagen fibres and increase in the mean number of the proliferating cells in the adrenal cortex.