ACKNOWLEDGEMENT AND DISCLAIMER: This is a draft chapter that has not been peer reviewed. While its production and figures were supported by the National Toxicology Program/National Institute of Environmental Health Sciences, it solely represents the opinion of the author, R. R. Maronpot.

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Adrenal Gland Figures 1-41

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Introduction

The mouse adrenal is comprised of a cortex and medulla that are embryologically distinctive in origin. While the adrenal cortex is essential for life, the function of the medulla is not essential for life. The mouse adrenal has several relatively unique species-specific histological features. There is a notable absence of a zona reticularis and a prominent x-zone at the cortico-medullary junction. Additionally, subcapsular proliferation of fusiform cells, frequent occurrence of accessory proliferative cortical nodules, and deposition of cortical lipogenic pigment are characteristics features of mouse adrenal glands. In contrast to the rat, mouse adrenal medullary tumors are morphologically comparable to human adrenal medullary tumors (Tischler et al., 1996). Among endocrine tissues, the adrenal is most commonly associated with chemically induced lesions but less so in the mouse compared to the rat. An understanding of the structure and function of the adrenal gland provides a sound basis for assessing and interpreting changes associated with exposure to xenobiotic agents and other challenges.

Embryology

(Figure 1)

The mouse adrenal is comprised of two tissue types that differ in embryological origin. The cortex is formed on gestation day 11 following condensation of celomic epithelium into the adrenogonadal primordium between the mesogastrium and urogenital fold. At the same gestational age, the medulla originates from neural crest ectoderm as two collections of sympathoblasts on opposites sides of the aorta (Sass 1996a). As the cortical anlage is being pushed between the mesonephros and aorta, the medullary sympathoblasts migrate along the sympathelic nerves toward the cortical anlage. On gestation day 12 the cortical anlage and medullary sympathoblasts are in close proximity and on gestation day 14 centrally located medullary cells are surrounded by cortical cells as both cell types undergo further cytoplasmic differentiation (Tischler & Sheldon 1996). Cortex and medulla development is nearly complete by gestation day 15 (Yarrington 1996). Medullary cells then enlarge with a chromaffin reaction evident by gestation day 16 (Sass 1996a). Mouse adrenal cortex and medulla are fully functional at birth. A cascade of transcription factors coordinate the cellular proliferative growth and functional development of the mouse adrenal gland among which steroidogenic factor 1 and GATA proteins as well as Sonic hedgehog (Shh) signaling have been identified in adrenal cortical development of the mouse (Kiiveri et al., 2001; Laufer et al., 2012; Walczak & Hammer 2015). Multiple endogenous and exogenous factors impact the highly orchestrated developmental growth and differentiation of the mouse adrenal cortex (Schulte et al., 2007). It has been postulated that the coordinated integration of these endogenous and exogenous events is channeled through a central integration involving ERK1/2 kinase activity (Hoeflich & Bielohuby 2009). Shh signals produced by partially differentiated cortical cells in the outer cortex/zona glomerulosa area are received by non-cortical subcapsular mesenchymal cells with both cellular populations having properties of adrenocortical progenitor or stem cells (Laufer et al., 2012). Using BrdU incorporation as a cellular growth marker, a scattered pattern of cellular proliferation is seen throughout the mouse adrenal cortex up to gestation day 13.5 while at later times BrdU-positive cells are localized in the subcapsular compartment, a potential stem cell location in the adult mouse adrenal cortex (Schulte et al., 2007).

Adrenal Gland Blood Supply

The dual embryological origins of the adrenal gland united under one capsule results in some important cross-talk between cortex and medulla that is mediated by the blood supply. The highly vascular adrenal gland receives its blood supply from several small branches of the dorsal aorta that form a subcapsular vascular plexus (Inomata and Sasano 2015). Separate branches from the subcapsular plexus provide arterial blood to the capsule, the cortex, and the medulla. The cortical arterial supply forms sinusoids that coalesce as a capillary network at the cortico-medullary junction. The dual blood supply to the medulla comes from the separate subcapsular plexus branch and from the cortico-medullary capillary network. The latter results in the medulla being exposed to corticosteroids necessary to activate PNMT and trigger a rapid stress response (Ehrhart-Bornstein and Bornstein 2008). Blood flow in the adrenal is centripetal with drainage into a central vein at the hilus. Description of innervation and vascular structures of the adrenal described in the 1934, 1936 and 1941 are nicely summarized by Thelma Dunn (Dunn 1970).

Anatomy and Histology

Adrenal Cortex

(Figures 2-5)

The adrenals are present bilaterally at the anterior poles of the right and left kidneys with the right adrenal closer to the kidney than the left adrenal. Female mouse adrenals are heavier and wider than male mouse adrenals and more translucent, presumably due to a higher lipid content (Yarrington 1996; Frith 1983; Frith & Dunn 1994; Tanaka & Matsuzawa 1995).

The mouse adrenal cortex consists of a zona glomerulosa, a zone fasciculata, and an x-zone. In contrast to most mammals, there is no clearly defined zona reticularis in the mouse adrenal cortex. The zona glomerulosa and zona fasiculata are distinguishable in the adult but often not clearly demarcated from one another. Zona glomerulosa cells are smaller, more basophilic, contain small uniform perinuclear lipid droplets, and form indistinct small arches compared to larger, more eosinophilic zona fasiculata cells that tend to form columns and usually have abundant cytoplasmic lipid droplets. The mouse has spindeloid cells that contain vitamin A scattered in the zona fasiculata (Kaufman et al., 2002). Ultrastructurally, zona glomerulosa cells contain round to ovoid mitochrondria with tubular cristae while zona fasiculata cells contain lipid droplets and abundant round mitochrondria (Kaufman et al., 2002).

X-zone

(Figures 5-8)

Scientific fascination with the x-zone continues today, 90 years after its initial description. This transitional region, unique to the mouse adrenal with as yet no clearly defined function, was first described by Masui and Tamura in 1927 which they considered to be the zona reticularis. Evelyn Howard-Miller (1927) at The Johns Hopkins University confirmed the Masui and Tamura findings in the newborn mouse adrenal which she named the x-zone and suggested it might be functionally homologous and histologically similar to a transitional zone in the human adrenal known as the fetal zone. Numerous early publications over the next four decades (cited by Sucheston and Cannon in 1972) investigated the function of this transitional zone in humans and various mammalian species (Ungar & Stabler 1980; Tsujio et al., 2009; Suto 2012; Deacon et al., 1986). In her paper, Martha Susheston compares the histological features of the transient-zone in human adrenals and the x-zone in the mouse and concludes that there is a difference in morphological development and probably function between these apparent homologous features. In the female mouse adrenal the x-zone zone is first seen at 10 to 14 days post-coitus with maximum development at and shortly after weaning when it can occupy greater than 50% of the cortex (Gersh & Grollman 1939; Howard-Miller 1927). It is noted that there are variations in when the x-zone is first present either during gestation or shortly after birth depending upon the specific report, suggesting that first appearance of an x-zone may be influenced by specific mouse strain. While descriptions vary with respect to tinctorial features of x-zone cells, they tend to be smaller and typically more basophilic compared to zona fasiculata cells (Starkey & Schmidt, Jr. 1938).

The natural history and age-sex dimorphism in the x-zone follows a progression of involution starting at 30 days of age in males with complete absence of the male x-zone at puberty. During this involution, the cells have condensed cytoplasm and pyknotic nuclei associated with hyperemia and are presumably carried away via the rich adrenal capillary and vascular network. This form of cell death is not accompanied by an inflammatory response and occurs without much evidence of classical apoptotic morphology even though apoptotic cell death is likely. The presence of a delicate fibrous capsule remaining at the corticomedullary junction is debatable. Gonadectomy preserves the x-zone in both males and females with some evidence of involution after 18 months (Deacon et al., 1986).

The x-zone in females continues to grow until 4 to 5 weeks of age when it represents a large part of the adrenal cortex. The natural history of x-zone involution in female mouse adrenal glands is protracted with mouse strain differences and evidence of tight controls involving multiple genes (Doughaday 1941; Suto 2012; Sucheston & Cannon 1972). In the non-parous female mouse, the x-zone persists for 100 to 200 days depending upon the specific strain. In the female DDD and JAX dba inbred mice, the x-zone persists for 200 days with subsequent involution accompanied by cytoplasmic vacuolization (Doughdady 1941; Suto 2012). In contrast, the involution in C57BL/6 females is complete at 100 days without accompanying cytoplasmic vacuolization. The dba x C57BL/6 hybrid x-zone involution resembles that of the dba parent. It is well established that the x-zone rapidly involutes with pregnancy starting 7 days after conception and with complete absence of this zone by gestation day 15 (Jones 1952). Regeneration of an x-zone following pregnancy and after castration in males was demonstrated in the early 1940s with the suggestion that the newly develop x-zone originated from the zona fasciculata (McPhail & Read 1942). Treatment of female mice with testosterone or progesterone also leads to complete involution of the x-zone (Jones 1952; Ungar & Stabler 1980). Based on multiple publications dated from 1927 to the present time, it is clear that the mouse adrenal x-zone is extremely sensitive to androgens and andromimetic hormonal effects, although that still leaves us without a clear understanding of its function. X-zone vacuolization and involution is apparently controlled by multiple genes with complex interactions (Suto 2012) but it is important to keep in mind that x-zone involution may occur without any evidence of cytoplasmic vacuolization.

Since the development and progressive involution of the x-zone is a normal process, one faces the choice of whether or not to document normally expected x-zone changes. In the past, diagnoses including degeneration, cytological vacuolization, fatty degeneration, regression, and atrophy have been used to document different stages of involution, implying a pathological process for this normal change. It is suggested that when evaluating an experimental or toxicity study, the normal age-associated process of x-zone involution need not require a diagnosis at all provided there is no difference between treated and control mice. For completeness, its presence could be described in the pathology narrative. In situations where there is a potential treatment effect, both “premature involution” and “x-zone persistence” are reasonable diagnostic terms that could be used along with assigning a severity score reflecting the degree of deviation from age-matched concurrent controls.

Medulla

(Figure 9)

The male and female mouse medulla occupies approximately 20% of the mouse adrenal volume and consists of irregular packets of polyhedral chromaffin cells and ganglion cells along with a rich vascular structure of venules and capillaries that is nicely summarized by Thelma Dunn (Dunn 1970). Chromaffin cells are basophilic with finely granular cytoplasm due to the presence of secretory granules. Based on staining reactions and ultrastructural features there are two types of chromaffin cells: epinephrine and norepinephrine secreting cells (Eranko 1955; Gorgas & Boch 1976; Tischler & Sheldon 1996). The cells closest to the cortex are norepinephrine secreting cells with the epinephrine secreting cells representing 75% of the medulla (Kaufman et al., 2002). Chromaffin cells secreting epinephrine have cytoplasmic granules 0.1 to 0.2 mm in diameter with moderately dense cores while nonepinephrine secreting cell granules have extremely dense cores. There is a minor population of small granule-containing chromaffin cells, scattered sustentacular cells, and sympathetic ganglion cells randomly distributed throughout the medulla. The medulla may extend to the adrenal capsular surface at the hilus. The organization and catecholamine synthetic function of the mouse adrenal medulla is under direct control of adrenocortical glucocorticoids and is indirectly affected by the beta-catenin pathway in cortical cells (Huang et al., 2012). The cortical to medullar blood flow pattern presumably delivers a high concentration of corticosterone to medullary cells as an endocrine stimulation and control mechanism.

Cell Proliferation & Adrenal Zonation

Because mammalian adrenocortical cells have a relatively low proliferative activity, investigations of the dynamic features of cell renewal in the cortex have been challenging, resulting in differing hypotheses of cell kinetics. Theories spanning years from 1883 to 1986 are briefly highlighted by investigators from Kyoto University (Kataoka et al., 1996). Theories include new cell origin from a stem cell compartment beneath the cortical capsule, at the interface between the zona glomerulosa and zona fasciculata with either unidirectional or bidirection mitration, cell origin in each adrenocortical zone independently, or all of these possibilities depending upon the interplay of numerous signals and factors (Walczak & Hammer 2015; Zajicek et al., 1986; Wright et al., 1973; Vinson 2016; Huang et al., 2010; McNicol & Duffy 1987; Jones 1948; Dunn 1970; Chang et al., 2013). Using pulse and flash [3H]thymidine labeling in 3- and 6-month old male ICR mice, the Kyoto University investigators concluded the adrenocortical cells proliferate at the border of the zona glomerulosa and zona fasciculata and move bidirectionally toward the cortical surface and the medulla (Kataoka et al., 1996). They conclude that almost all cortical cells are replaced by renewed cells within 200 days.

Unidirectional centripetal zonal migration of adrenocortical cells was first proposed in the early 1930’s primarily using rats (Jones 1948). The majority of studies addressing centripetal migration of dividing adrenocortical cells have been done using rats and different means of labeling proliferating cells using BrdU and thymidine labeling McNicol, 1987; Bertholet 1980). Movement of thymidine labeled cells from the cortex to the medulla took 104 days in rats (Zajicek et al., 1986). In a study of chronic stress caused by immobilizing rats, there was a decreased cell proliferation in the adrenal cortex but the authors point out that their findings are consistent with the centripetal migration theory (Bozzo et al., 2011). Using pulse labeling in adult F1 mice (C57BL/6 x CBA/Ca), BrdU-labeled cells moved centripetally 13-20 micrometers per day toward the medulla with some outer cortex cells retaining their label for 13-23 weeks in pulse-chase experiments (Chang et al., 2013).

A 2015 review paper investigates regulatory factors & their signaling associated with stem cell niches in the mammalian adrenal cortex with discussion of implications for disease (Walczak & Hammer 2015). The authors point out that the presence of a stem cell compartment is consistent with cellular proliferation needed to maintain adult adrenal volume and function. Under homeostatic conditions, a slowly cycling capsular/subcapsular stem cell population with centripetal migration permits replenishment of the adrenal cortex. A family of secreted Hedgehog molecules participate in a complex paracrine and endocrine regulatory cascade including SF-1, Wnt, IGF, FGF, angiotensin II and ACTH leading to the activation of the stem cell pool thus allowing for a proliferative response with subsequent differentiation into steroidogenic cells. Adrenal insufficiency diseases can be a consequence of loss or gain of function of genes and their associated transcription factors and signalling pathways perturbing maintenance and function of adrenocortical stem and progenitor cell compartments.

Fully differentiated epinephrine (E) and norepinephrine (NE) medullary cells are capable of replication (Jurecka et al., 1978; Tischler and Sheldon, 1996). Based on BrdU-staining, the basal chromaffin cell labeling index in the adult mouse is approximately 1% with only marginal differences between different mouse strains (Tischer et al., 1997). C57BL/6 male chromaffin cells do respond to reserpine administration with a one-week labeling index of 10.8% versus 4.4% in concurrent controls. It has been suggested that the intrinsically lower proliferative capacity of mouse chromaffin cells compared to rat chromaffin cells may be related to the lower incidence of pheochromocytomas in mice
than in rats.

Adrenal Function

Adrenal Cortex

The cortex is a major site of steroidogenesis with synthesis and secretion of hormones regulated by the hypothalamic-anterior pituitary-adrenal (HPA) axis, the renin-angiotensin system, and other factors. Cholesterol is the precursor for steroid hormone production with conversion mediated by CYP11A1 under ACTH influence. Steroid hormone secretion is circadian and immediate with blood levels reflecting the rate of synthesis. Fetal and neonatal steroidogenesis undergoes dynamic age-associated changes with early appearance of functional glucocorticoid receptors and enzymatic activity, and early dynamic changes in glucocorticoid levels in fetal tissue with increased glucocorticoid levels during growth to adulthood (Yarrington 1996). The mouse adrenal cortex mediates stress responses by secretion of mineralocorticoids (aldosterone) by the zona glomerulosa and glucocorticoids by the zona fasciculata. Zona glomerulosa cells may also exhibit some neuroendocrine properties (Ehrhart-Bornstein & Hilbers 1998). In mice the principal glucocorticoid is costicosterone. Circulating costicosterone levels, adrenal gland weight, and volume of the zona fasiculata are higher in females than in males (Bielohuby et al., 2007).

Adrenocortical steroidogenesis is dependent upon pituitary ACTH, hormone receptor interactions, neurotransmitters, cytokines and growth factor networks and is mediated by direct cell-cell communication of regulatory molecules via gap junctions (Bielohuby et al., 2007; Bell & Murray 2016). There are two sources of the starting material, cholesterol: extracellular cholesterol from the diet or synthesized in the liver and intracellular cholesterol synthesized de novo in adrenal cortical cells from acetate (Yarrington 1996). The steroidogenesis cascade starts when cholesterol is esterfied with fatty acids and stored within adrenal cortical cells in lipid droplets. Free cholesterol that is released from adrenal cortical cytoplasmic lipid droplets following hormone stimulation is the preferred source of the cholesterol substrate for steroidogenesis (Shen et al., 2016). Once steroidogenesis starts the cholesterol is degraded by esterase and translocated to mitochondria where it is hydroxylated to form pregnenolone. From the mitochondrial space pregnenolone is shuttled to the endoplasmic reticulum and is enzymatically converted to 11-deoxycorticosterone and sent back to mitochondrial space for 11-hydroxylation to form corticosterone. [Note: Unlike some other species, the mouse produces corticosterone rather than cortisole.] Following a hydroxylase and a subsequent dehydrogenase step, corticosterone is converted to aldosterone.

Adrenal mineralocorticoid and glucocorticoids are not stored but enter the blood stream directly upon synthesis. Corticosterone secretion is primarily governed by the hypothalamic-pituitary-adrenal axis and feedback mechanism with stimulation of secretion in response to ACTH. Under normal physiological conditions the circadian levels of corticosterone are lowest at mid-day and highest shortly before midnight (Yarrington 1996). As mice age there is a decrease in circulating corticosterone as well as decreased responsiveness to ACTH.

Aldosterone secretion is also responsive to ACTH but also is influenced by angiotensin II acting on non-ACTH receptors in the zona glomerulosa. Aldosterone acts on the renal distal convoluted tubules to conserve sodium and excrete potassium. Increases in serum potassium trigger renin in the renal juxtaglomerular apparatus to react with plasma angiotensinogen to form angiotensin I that then undergoes enzymatic conversion to its active form, angiotensin II. Angiotensin II can act as endocrine, autocrine/paracrine and intracrine hormone. In the adrenal zona glomerulosa it acts on non-ACTH receptors to stimulate the release of aldosterone.

Aldosterone is the primary mineralocorticoid of the adrenal cortex and is produced by the action of aldosterone synthase (CYP11B2). Aldosterone is responsible for long-term maintenance of blood pressure and achieves this by promoting reabsorption of sodium and increased secretion of potassium and hydrogen ions in the distal convoluted tubules and collecting ducts of the kidneys. An innate electrical excitability of mouse zona glomerulosa cells provides a recurrent Ca2+ channel necessary for sustained production of aldosterone with regulation by potassium and the angiotensin-renin system located in the juxtaglomerular cells of the kidneys (Hu et al., 2016).. Insufficient production of aldosterone can lead to reduced extracellular fluid volume and death from hypovolemic shock.

The glucocorticoids such as cortisol (cortocosterone in mice) are secreted primarily by the zona fasciculata and effects include mobilization of fats, carbohydrates and proteins in addition to enhancing the activity of other hormones, including glucagon and catecholamines. Following binding to appropriate targets, glucocorticoid effects include release of amino acids from the body, stimulation of lipolysis and gluconeogenesis, increasing water retention, and strengthening cardiac muscular contraction in addition to having anti-inflammatory effects. Regulation of synthesis and release of corticosterone in mice is mediated by adrenocorticotropic hormone (ACTH) from the pituitary and controlled by a feed-back mechanism. Under normal conditions the zona fasciculata produces a basal level of glucocorticoid but can produce an enhanced burst of hormone in response to ACTH secretion.

Adrenal Medulla

Medullary cells are basically modified postganglionic cells of the autonomic nervous system and as a cluster can be consider a ganglion of the sympathetic nervous system. The autonomic nervous system exerts direct control over the medullary chromaffin cells, the principal site where tyrosine is converted into the catecholamines epinephrine, norepinephrine, and dopamine. Catecholamine synthesis in the medulla is stimulated by paracrine action of adrenocortical glucocorticoids and the beta-catenin pathway in adrenal cortical cells has an indirect role in the differentiation and proper organization of the adrenal medulla (Huang et al., 2012). Hormone release can occur quickly in response to stressors (fight or flight response) and effects include increased heart rate and cardiac contraction, smooth muscle dilation, and increase of glycogenolysis and fatty acid release from adipose tissue. The adrenal medulla is not essential for life.

Tissue Changes and Lesions

Adrenal Cortex

Accessory Cortical Nodules

(Figures 10-12)

Accessory adrenal cortical nodules represent the primary congenital lesion in mice, are relatively common with up to a 50% incidence in some strains, and are formed by growth of detached coelomic epithelial primordia during development of the adrenal cortex (Sass 1996b; Hummel 1958; Jayne 1963). These nodules are commonly located at one pole of the adrenal gland in contact with the adrenal capsule but can also occur separate from the adrenal in periadrenal adipose tissue. They are not clinically or toxicologically significant but it is noted that toxic and proliferative changes in the adrenal cortex may also be seen in these accessory nodules.

Katharine Hummel (1958) mentioned older literature describing accessory cortical adrenal tissue in mice and then provided data on adrenals from over 4000 mice representing 9 mouse strains and hybrids maintained at the Jackson Laboratory. She found discrete accessory cortical nodules in all strains and hybrids and described the nodules as comprised of cells arranged in layers surrounding a central blood vessel and resembling a normal adrenal with typical cortical layers but noted the absence of a medulla. Accessory nodules were more prevalent in females, were typically unilateral on the left side and multiple nodules were present in most mouse strains. These nodules developed the same aging changes as the normal adrenal cortex, including development of subcapsular spindle cell hyperplasia. While an embryological origin is most likely for occurrence of accessory cortical nodules, based on older literature cited by Hummel plus her own observations, an adult origin for these nodules can’t be excluded. A useful graphical representation of the inbred strain distribution of accessory cortical nodules is provided in the book chapter by Bernard Sass (Sass 1996b).

Waring and Scott (1937) have provided an extensive description of accessory cortical nodules and describe an accessory adrenal gland in mouse (sex and strain not identified). The double adrenal gland consisted of a large normal adrenal with a smaller adrenal fused to and protruding from the larger adrenal. Both adrenals had a complete cortex and medulla.

Adrenal cortical nodules are round to ovoid and surrounded by a thin fibrous capsule with recognizable zona glomerulosa and zone fasciculata cells. They can develop the same degenerative and proliferative changes as the adrenal cortex proper and it is recommended that they be diagnosed without a severity grade when present (https://ntp.niehs.nih.gov/nnl/).

Cytoplasmic Alteration

Cytoplasmic alteration is a generic diagnostic category that is preferred by some pathologists as representative of a spectrum of cortical morphological changes including cytoplasmic vacuolation and focal and diffuse cellular hypertrophy. When using non-specific diagnoses such as cellular alteration, it is recommended that a definitive description of the cellular alteration be provided in the pathology narrative.

Cortical Vacuolation

In normal unstressed mice, adrenocortical cell cytoplasm may appear vacuolated because of lipid representing cholesterol acetate and steroid precursors. Adrenal cortical vacuolation occurs to varying degrees as part of the diurnal functional activity of the cortical cells but can increase secondary to disruption of normal steroidogenesis (Rosol et al., 2001; Greaves 2012; https://ntp.niehs.nih.gov/nnl/index.htm). Cytochrome-450 in the adrenal cortex is involved in the biotransformation of cholesterol into steroid hormones and any disruption of CYP action can cause a diffuse increase in cytoplasmic lipid droplets in the zona fasciculata. Disruption can be a consequence of xenobiotic inactivatio