Adrenal Cortex Development and Maintenance: Knowledge Acquired From Mouse Models.

The adrenal cortex is an endocrine organ organized into concentric zones that are specialized to produce specific steroid hormones essential for life. The development and maintenance of the adrenal cortex are complex, as a fetal adrenal is first formed from a common primordium with the gonads, followed by its separation in a distinct primordium, the invasion of the adrenal primordium by neural crest-derived cells to form the medulla, and finally its encapsulation. The fetal cortex is then replaced by a definitive cortex, which will establish zonation and be maintained throughout life by regeneration relying on the proliferation, centripetal migration, and differentiation of several stem/progenitor cell populations whose activities are sex-specific. Here, we highlight the advances made, using transgenic mouse models, to delineate the molecular mechanisms regulating these processes.

Adrenal medulla development and medullary-cortical interactions.

The mammalian adrenal gland is composed of two distinct tissue types in a bidirectional connection, the catecholamine-producing medulla derived from the neural crest and the mesoderm-derived cortex producing steroids. The medulla mainly consists of chromaffin cells derived from multipotent nerve-associated descendants of Schwann cell precursors. Already during adrenal organogenesis, close interactions between cortex and medulla are necessary for proper differentiation and morphogenesis of the gland. Moreover, communication between the cortex and the medulla ensures a regular function of the adult adrenal. In tumor development, interfaces between the two parts are also common. Here, we summarize the development of the mammalian adrenal medulla and the current understanding of the cortical-medullary interactions under development and in health and disease.

Triphenyltin chloride reduces the development of rat adrenal cortex during puberty.

Triphenyltin has been classified as an endocrine disruptor. However, whether triphenyltin interferes with the adrenal glands during puberty remains unknown. Here, we reported the effects of triphenyltin on the adrenal glands in rats. Male Sprague Dawley rats (age of 35 days) were orally administered with 0, 0.5, 1, or 2 mg/kg/day triphenyltin for 18 days. Triphenyltin significantly lowered corticosterone levels at 1 and 2 mg/kg and adrenocorticotropic hormone at 2 mg/kg. The RNA-Seq analysis detected multiple differentially expressed genes. Four down-regulated genes were transcription factor genes (Nr4a1, Nr4a2, Nr4a3, and Ppard), which might be associated with the suppression of the adrenal cortex function. RNA-seq and qPCR showed that triphenyltin dose-dependently down-regulated the expression of the genes for cholesterol transport and biosynthesis, including Scarb1, Ldlr, Hmgcs1, Hmgcr, and Hsd17b7. Further Western blotting revealed that it lowered NR4A1, PPRAD, LDLR, and HMGCS1 protein levels. We treated H295R adrenal cells with 1-100 nM triphenyltin for 72 h. Triphenyltin induced significant higher ROS production at 100 nM and did not induce apoptosis at 10 and 100 nM. In conclusion, triphenyltin inhibits production of corticosterone via blocking the expression of cholesterol uptake transporters and cholesterol biosynthesis.

Embryonic Development and Adult Regeneration of the Adrenal Gland.

The adrenal gland plays a pivotal role in an organism's health span by controlling the endocrine system. Decades of research on the adrenal gland have provided multiscale insights into the development and maintenance of this essential organ. A particularly interesting finding is that founder stem/progenitor cells participate in adrenocortical development and enable the adult adrenal cortex to regenerate itself in response to hormonal stress and injury. Since major advances have been made in understanding the dynamics of the developmental process and the remarkable regenerative capacity of the adrenal gland, understanding the mechanisms underlying adrenal development, maintenance, and regeneration will be of interest to basic and clinical researchers. Here, we introduce the developmental processes of the adrenal gland and discuss current knowledge regarding stem/progenitor cells that regulate adrenal cortex remodeling and regeneration. This review will provide insights into the fascinating ongoing research on the development and regeneration of the adrenal cortex.

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.

A ZNRF3-dependent Wnt/ß-catenin signaling gradient is required for adrenal homeostasis.

Spatiotemporal control of Wnt signaling is essential for the development and homeostasis of many tissues. The transmembrane E3 ubiquitin ligases ZNRF3 (zinc and ring finger 3) and RNF43 (ring finger protein 43) antagonize Wnt signaling by promoting degradation of frizzled receptors. ZNRF3 and RNF43 are frequently inactivated in human cancer, but the molecular and therapeutic implications remain unclear. Here, we demonstrate that adrenocortical-specific loss of ZNRF3, but not RNF43, results in adrenal hyperplasia that depends on Porcupine-mediated Wnt ligand secretion. Furthermore, we discovered a Wnt/ß-catenin signaling gradient in the adrenal cortex that is disrupted upon loss of ZNRF3. Unlike ß-catenin gain-of-function models, which induce high Wnt/ß-catenin activation and expansion of the peripheral cortex, ZNRF3 loss triggers activation of moderate-level Wnt/ß-catenin signaling that drives proliferative expansion of only the histologically and functionally distinct inner cortex. Genetically reducing ß-catenin dosage significantly reverses the ZNRF3-deficient phenotype. Thus, homeostatic maintenance of the adrenal cortex is dependent on varying levels of Wnt/ß-catenin activation, which is regulated by ZNRF3.

The human adrenal cortex: growth control and disorders.

This review summarizes key knowledge regarding the development, growth, and growth disorders of the adrenal cortex from a molecular perspective. The adrenal gland consists of two distinct regions: the cortex and the medulla. During embryological development and transition to the adult adrenal gland, the adrenal cortex acquires three different structural and functional zones. Significant progress has been made in understanding the signaling and molecules involved during adrenal cortex zonation. Equally significant is the knowledge obtained regarding the action of peptide factors involved in the maintenance of zonation of the adrenal cortex, such as peptides derived from proopiomelanocortin processing, adrenocorticotropin and N-terminal proopiomelanocortin. Findings regarding the development, maintenance and growth of the adrenal cortex and the molecular factors involved has improved the scientific understanding of disorders that affect adrenal cortex growth. Hypoplasia, hyperplasia and adrenocortical tumors, including adult and pediatric adrenocortical adenomas and carcinomas, are described together with findings regarding molecular and pathway alterations. Comprehensive genomic analyses of adrenocortical tumors have shown gene expression profiles associated with malignancy as well as methylation alterations and the involvement of miRNAs. These findings provide a new perspective on the diagnosis, therapeutic possibilities and prognosis of adrenocortical disorders.

Adrenocortical development: Lessons from mouse models.

The adrenocortical gland undergoes structural and functional remodelling in the fetal and postnatal periods. After birth, the fetal zone of the gland undergoes rapid involution in favor of the definitive cortex, which reaches maturity with the emergence of the zona reticularis(zR) at the adrenarche. The mechanisms underlying the adrenarche, the process leading to pre-puberty elevation of plasma androgens in higher primates, remain unknown, largely due to lack of any experimental model. By following up fetal and definitive cortex cell lines in mice, we showed that activation of protein kinase A (PKA) signaling mainly impacts the adult cortex by stimulating centripetal regeneration, with differentiation and then conversion of the zona fasciculata into a functional zR. Animals developed Cushing syndrome associated with primary hyperaldosteronism, suggesting possible coexistence of these hypersecretions in certain patients. Remarkably, all of these traits were sex-dependent: testicular androgens promoted WNT signaling antagonism on PKA, slowing cortical renewal and delaying onset of Cushing syndrome and the establishment of the zR in male mice, this being corrected by orchidectomy. In conclusion, zR derives from centripetal conversion of the zona fasciculata under cellular renewal induced by PKA signaling, determining the size of the adult cortex. Finally, we demonstrated that this PKA-dependent mobilization of cortical progenitors is sexually dimorphic and could, if confirmed in humans, account for female preponderance in adrenocortical pathologies.

The human adrenal cortex: growth control and disorders

This review summarizes key knowledge regarding the development, growth, and growth disorders of the adrenal cortex from a molecular perspective. The adrenal gland consists of two distinct regions: the cortex and the medulla. During embryological development and transition to the adult adrenal gland, the adrenal cortex acquires three different structural and functional zones. Significant progress has been made in understanding the signaling and molecules involved during adrenal cortex zonation. Equally significant is the knowledge obtained regarding the action of peptide factors involved in the maintenance of zonation of the adrenal cortex, such as peptides derived from proopiomelanocortin processing, adrenocorticotropin and N-terminal proopiomelanocortin. Findings regarding the development, maintenance and growth of the adrenal cortex and the molecular factors involved has improved the scientific understanding of disorders that affect adrenal cortex growth. Hypoplasia, hyperplasia and adrenocortical tumors, including adult and pediatric adrenocortical adenomas and carcinomas, are described together with findings regarding molecular and pathway alterations. Comprehensive genomic analyses of adrenocortical tumors have shown gene expression profiles associated with malignancy as well as methylation alterations and the involvement of miRNAs. These findings provide a new perspective on the diagnosis, therapeutic possibilities and prognosis of adrenocortical disorders.

Kisspeptin Is a Novel Regulator of Human Fetal Adrenocortical Development and Function: A Finding With Important Implications for the Human Fetoplacental Unit.

Context: The human fetal adrenal (HFA) is an integral component of the fetoplacental unit and important for the maintenance of pregnancy. Low kisspeptin levels during pregnancy are associated with miscarriage, and kisspeptin and its receptor are expressed in the HFA. However, the role of kisspeptin in fetal adrenal function remains unknown. Objective: To determine the role of kisspeptin in the developing HFA. Design: Experiments using H295R and primary HFA cells as in vitro models of the fetal adrenal. Association of plasma kisspeptin levels with HFA size in a longitudinal clinical study. Setting: Academic research center and tertiary fetal medicine unit. Participants: Thirty-three healthy pregnant women were recruited at their 12-week routine antenatal ultrasound scan. Main Outcome Measures: The spatiotemporal expression of Kiss1R in the HFA. The production of dehydroepiandrosterone sulfate (DHEAS) from HFA cells after kisspeptin treatment, alone or in combination with adrenocorticotropic hormone or corticotropin-releasing hormone. Fetal adrenal volume (FAV) and kisspeptin levels at four antenatal visits (∼20, 28, 34, and 38 weeks' gestation). Results: Expression of Kiss1R was present in the HFA from 8 weeks after conception to term and was shown in the inner fetal zone. Kisspeptin significantly increased DHEAS production in H295R and second-trimester HFA cells. Serial measurements of kisspeptin confirmed a correlation with FAV growth in the second trimester, independent of sex or estimated fetal weight. Conclusions: Kisspeptin plays a key role in the regulation of the HFA and thus the fetoplacental unit, particularly in the second trimester of pregnancy.

Targeted Disruption of YAP and TAZ Impairs the Maintenance of the Adrenal Cortex.

Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) are functionally redundant transcriptional regulators that are downstream effectors of the Hippo signaling pathway. They act as major regulators of stem cell maintenance, cell growth, and differentiation. To characterize their roles in the adrenal cortex, we generated a mouse model in which Yap and Taz were conditionally deleted in steroidogenic cells (Yapflox/flox;Tazflox/flox;Nr5a1cre/+). Male Yapflox/flox;Tazflox/flox;Nr5a1cre/+ mice were characterized by an age-dependent degeneration of the adrenal cortex associated with an increase in apoptosis and a progressive reduction in the expression levels of steroidogenic genes. Evaluation of the expression levels of stem and progenitor cell population markers in the adrenal glands of Yapflox/flox;Tazflox/flox;Nr5a1cre/+ mice also showed the downregulation of sonic hedgehog (Shh), a marker of the subcapsular progenitor cell population. Gross degenerative changes were not observed in the adrenal glands of Yapflox/flox;Tazflox/flox;Nr5a1cre/+ females, although steroidogenic capacity and Shh expression were reduced, suggesting that mechanisms of adrenocortical maintenance are sex specific. These results define a crucial role for YAP and TAZ in the maintenance of the postnatal adrenal cortex.

Genetics of Adrenocortical Development and Tumors.

This article links the understanding of developmental physiology of the adrenal cortex to adrenocortical tumor formation. Many molecular mechanisms that lead to formation of adrenocortical tumors have been discovered via next-generation sequencing approaches. The most frequently mutated genes in adrenocortical tumors are also factors in normal adrenal development and homeostasis, including those that alter the p53 and Wnt/ß-catenin pathways. In addition, dysregulated protein kinase A signaling and ARMC5 mutations have been identified as key mediators of adrenocortical tumorigenesis. The growing understanding of genetic changes that orchestrate adrenocortical development and disease pave the way for potential targeted treatment strategies.

Aging-related impairment of urine-concentrating mechanisms correlates with dysregulation of adrenocortical angiotensin type 1 receptors in male Fischer rats.

To investigate age-associated impairments in fluid homeostasis, 4-mo (young) and 32-mo (old) Fischer 344/BN male rats were studied before and after a dietary sodium load. Transferring young rats from a low-sodium (LS) to a high-sodium (HS) diet increased water intake and urine volume by 1.9- and 3.0-fold, respectively, while urine osmolality and plasma aldosterone decreased by 33 and 98%. Concomitantly, adrenocortical angiotensin type 1 receptor (AT1R) density decreased by 35%, and AT1bR mRNA decreased by 39%; no changes were observed in AT1aR mRNA. In contrast, the increase in water intake (1.4-fold) was lower in the old rats, and there was no effect of the HS diet on urine volume or urine osmolality. AT1R densities were 29% less in the old rats before transferring to the HS diet, and AT1R densities were not reduced as rapidly in response to a HS diet compared with the young animals. After 6 days on the HS diet, plasma potassium was lowered by 26% in the old rats, whereas no change was detected in the young rats. Furthermore, while plasma aldosterone was substantially decreased after 2 days on the HS diet in both young and old rats, plasma aldosterone was significantly lower in the old compared with the young animals after 2 wk on the LS diet. These findings suggest that aging attenuates the responsiveness of the adrenocortical AT1R to a sodium load through impaired regulation of AT1bR mRNA, and that this dysregulation contributes to the defects in water and electrolyte homeostasis observed in aging.

Expression of estrogen, estrogen related and androgen receptors in adrenal cortex of intact adult male and female rats.

INTRODUCTION: Adrenocortical activity in various species is sensitive to androgens and estrogens. They may affect adrenal cortex growth and functioning either via central pathways (CRH and ACTH) or directly, via specific receptors expressed in the cortex and/or by interfering with adrenocortical enzymes, among them those involved in steroidogenesis. Only limited data on expression of androgen and estrogen receptors in adrenal glands are available. Therefore the present study aimed to characterize, at the level of mRNA, expression of these receptors in specific components of adrenal cortex of intact adult male and female rats. MATERIAL AND METHODS: Studies were performed on adult male and female (estrus) Wistar rats. Total RNA was isolated from adrenal zona glomerulosa (ZG) and fasciculate/reticularis (ZF/R). Expression of genes were evaluated by means of Affymetrix® Rat Gene 1.1 ST Array Strip and QPCR. RESULTS: By means of Affymetrix® Rat Gene 1.1 ST Array we examined adrenocortical sex differences in the expression of nearly 30,000 genes. All data were analyzed in relation to the adrenals of the male rats. 32 genes were differentially expressed in ZG, and 233 genes in ZF/R. In the ZG expression levels of 24 genes were lower and 8 higher in female rats. The more distinct sex differences were observed in the ZF/R, in which expression levels of 146 genes were lower and 87 genes higher in female rats. Performed analyses did not reveal sex differences in the expression levels of both androgen (AR) and estrogen (ER) receptor genes in the adrenal cortex of male and female rats. Therefore matrix data were validated by QPCR. QPCR revealed higher expression levels of AR gene both in ZG and ZF/R of male than female rats. On the other hand, QPCR did not reveal sex-related differences in the expression levels of ERα, ERß and non-genomic GPR30 (GPER-1) receptor. Of those genes expression levels of ERα genes were the highest. In studied adrenal samples the relative expression of ERα mRNA was higher than ERß mRNA. In adrenals of adult male and female rats expression levels of estrogen-related receptors ERRα and ERRß were similar, and only in the ZF/R of female rats ERRγ expression levels were significantly higher than in males. We also analyzed expression profile of three isoforms of steroid 5α-reductase (Srd5a1, Srd5a2 and Srd5a3) and aromatase (Cyp19a1) and expression levels of all these genes were similar in ZG and ZF/R of male and female rats. CONCLUSIONS: In contrast to Affymetrix microarray data QPCR revealed higher expression levels of AR gene in adrenal glands of the male rats. In adrenals of both sexes expression levels of ERa, ERb, non-genomic GPR30 (GPER-1), ERR α and ERRß receptors were comparable. The obtained results suggest that acute steroidogenic effect of estrogens on corticosteroid secretion may be mediated by non-genomic GPR30.

Development of adrenal cortex zonation.

The human adult adrenal cortex is composed of the zona glomerulosa (zG), zona fasciculata (zF), and zona reticularis (zR), which are responsible for production of mineralocorticoids, glucocorticoids, and adrenal androgens, respectively. The final completion of cortical zonation in humans does not occur until puberty with the establishment of the zR and its production of adrenal androgens; a process called adrenarche. The maintenance of the adrenal cortex involves the centripetal displacement and differentiation of peripheral Sonic hedgehog-positive progenitors cells into zG cells that later transition to zF cells and subsequently zR cells.

Adrenocortical growth and cancer.

The adrenal gland consists of two distinct parts, the cortex and the medulla. Molecular mechanisms controlling differentiation and growth of the adrenal gland have been studied in detail using mouse models. Knowledge also came from investigations of genetic disorders altering adrenal development and/or function. During embryonic development, the adrenal cortex acquires a structural and functional zonation in which the adrenal cortex is divided into three different steroidogenic zones. Significant progress has been made in understanding adrenal zonation. Recent lineage tracing experiments have accumulated evidence for a centripetal differentiation of adrenocortical cells from the subcapsular area to the inner part of the adrenal cortex. Understanding of the mechanism of adrenocortical cancer (ACC) development was stimulated by knowledge of adrenal gland development. ACC is a rare cancer with a very poor overall prognosis. Abnormal activation of the Wnt/ß-catenin as well as the IGF2 signaling plays an important role in ACC development. Studies examining rare genetic syndromes responsible for familial ACT have played an important role in identifying genetic alterations in these tumors (like TP53 or CTNNB1 mutations as well as IGF2 overexpression). Recently, genomic analyses of ACT have shown gene expression profiles associated with malignancy as well as chromosomal and methylation alterations in ACT and exome sequencing allowed to describe the mutational landscape of these tumors. This progress leads to a new classification of these tumors, opening new perspectives for the diagnosis and prognostication of ACT. This review summarizes current knowledge of adrenocortical development, growth, and tumorigenesis.

Regulation of the adrenocortical stem cell niche: implications for disease.

Stem cells are endowed with the potential for self-renewal and multipotency. Pluripotent embryonic stem cells have an early role in the formation of the three germ layers (ectoderm, mesoderm and endoderm), whereas adult tissue stem cells and progenitor cells are critical mediators of organ homeostasis. The adrenal cortex is an exceptionally dynamic endocrine organ that is homeostatically maintained by paracrine and endocrine signals throughout postnatal life. In the past decade, much has been learned about the stem and progenitor cells of the adrenal cortex and the multiple roles that these cell populations have in normal development and homeostasis of the adrenal gland and in adrenal diseases. In this Review, we discuss the evidence for the presence of adrenocortical stem cells, as well as the various signalling molecules and transcriptional networks that are critical for the embryological establishment and postnatal maintenance of this vital population of cells. The implications of these pathways and cells in the pathophysiology of disease are also addressed.

3ßHSD and CYB5A double positive adrenocortical cells during adrenal development/aging.

Androstenedione is a common precursor of sex steroids produced and secreted in the human adrenal gland and produced by 3ß-hydroxysteroid dehydrogenase (3ßHSD), 17ß-hydroxylase/17,20-lyase (CYP17) and cytochrome b5 (CYB5A). 3ßHSD is expressed in the zona glomerulosa (ZG) and fasciculata (ZF), CYP17 in the ZF and zona reticularis (ZR) and CYB5A in the ZR, respectively. We previously demonstrated the presence of cortical parenchymal cells co-expressing 3ßHSD and CYB5A with hybrid features of both ZF and ZR in human adrenal cortex and hypothesized that these cells may play an important role in androstenedione production in human adrenal gland. Age-related morphologic development of these hybrid cells has, however, not been studied. Therefore, in this study, 48 human adrenal specimens from various age groups were retrieved. Double-immunohistochemical analyses were used in order to study the correlation between this hybrid cell type and age. In both male and female adrenal cortex, the means of total adrenocortical area, the area positive for CYB5A and its ratio reached highest peak in the 21-40-year-old (y.o.) group. The greatest overlap between 3ßHSD and CYB5A in both total and relative area was present in the 13-20 y.o. group. For all the markers mentioned above, statistically significant differences were detected among the different age groups examined (p < 0.05). These findings indicated that both area and ratio of 3ßHSD and CYB5A double positive cells, which could represent the hybrid cells of ZF and ZR, are correlated with human adrenal development and could subsequently influence age-related serum androstenedione levels.

The presentation of adrenarche is sexually dimorphic and modified by body adiposity.

CONTEXT: Adrenarche refers to the onset of increased production of adrenal androgens in childhood leading variably to clinical signs of androgen action. The prevalence and presentation of adrenarche in prepubertal girls and boys is not well known. OBJECTIVE: Our objective was to examine the prevalence and clinical presentation of adrenarche in a population sample of prepubertal children aged less than 9 years. DESIGN AND PARTICIPANTS: This cross-sectional study included prepubertal children (209 girls and 228 boys; median age 7.6 [range 6.8-8.9] years) taking part in The Physical Activity and Nutrition in Children (PANIC) Study. MAIN OUTCOME MEASURE: The prevalence of adrenarche was assessed. RESULTS: The prevalence of any clinical sign of androgen action was higher in girls than in boys (26.1% vs 10.0%; P < .001) and biochemical adrenarche without any clinical sign was less common in girls than in boys (8.1% vs 16.7%; P = .007). When premature adrenarche was defined by serum dehydroepiandrosterone sulfate concentration ≥1 µmol/L (≥37 µg/dL) and any clinical sign before the age of 8 years in girls and 9 years in boys, its total prevalence was 8.6% in girls and 1.8% in boys. The risk of having any clinical sign increased with higher body fat percentage in boys and with higher serum dehydroepiandrosterone sulfate concentration in girls. CONCLUSIONS: Clinical signs of androgen action are more common, but biochemical adrenarche without any clinical sign is less common in prepubertal girls than boys. This sexual dimorphism of adrenarche might be explained by sex-dependent differences in peripheral androgen metabolism or action that are modified by body fat.