ACKNOWLEDGEMENT AND DISCLAIMER: This is a draft chapter that has not been peer reviewed.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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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 inactivation of CYP-450 enzymes or perturbation of the HPA axis resulting in increased ACTH secretion leading to secondary increase in cytoplasmic lipid. Additionally, adrenal CYP-450 enzymes can activate xenobiotics leading to reactive oxygen species that damage cell membranes causing accumulation of intracytoplasmic phospholipids.

Increased cortical vacuolation occurs at low incidence in male and female CD-1 mice (Petterino et al., 2015). A diagnosis of increased cortical vacuolation should be reserved for those instances where it is clearly in excess of what is seen in concurrent and historical controls. A treatment-related decrease in cortical vacuolation versus concurrent controls can occur from shutdown of steroidogenesis or perturbation of ability to store cholesterol acetate and steroid precursors.

Cortical Hypertrophy

(Figures 13-17)

Cortical hypertrophy, as distinct from cortical hyperplasia, can be diffuse or focal. Discrete rounded focal aggregates of exclusively hypertrophic cells can occur and are readily identified because they are tinctorially distinct from the adjacent zona fasciculata cells. The morphological features in hypertrophic cells can be quite variable with clear, homogeneously eosinophilic, vacuolated or a mixture of these cytoplasmic features. In the mouse, because a clear distinction between focal cortical vacuolation and focal cortical hypertrophy cannot be easily made, the recommended default diagnosis is hypertrophy with the option of describing the cytological details in the pathology narrative. Depending upon the degree of hypertrophy, there may be minimal compression of adjacent zona fasiculata parenchyma and cells in these lesions may have degenerative changes (https://ntp.niehs.nih.gov/nnl/endocrine/adrenal/cxvacuol/index.htm). In older B6C3F1 mice focal hypertrophy of the zona fasiculata is more common than focal hyperplasia and may extend into the zona glomerulosa. Depending upon the degree of focal hypertrophy, there may be minimal compression of adjacent zona fasiculata parenchyma. In hypertrophic lesions, decreased nuclear density is apparent because of the increased cytoplasmic size. While the functional significance of diffuse hypertrophy may result from increased storage or failure to release steroid precursors, the functional significance of focal hypertrophy is uncertain. Bilateral occurrence of cortical hypertrophy suggests a systemic effect or response.

Amyloidosis

(Figure 18)

Amyloidosis is the result of the tissue buildup of insoluble hydrophobic fibrils of misfolded proteins called amyloids. Amyloidosis in mice is typically a systemic condition with intercellular deposits of homogeneous light staining eosinophilic material in multiple organs including kidneys, liver, intestine and genital tissues. It is strain- and age-dependent, more frequent in females than in males, and is common finding in aged mice, including A/J, C57BL/6, C3H, CBA strains (Chandra et al., 2013; Sass 1996c). Incidences as high as 100% have been reported in some inbred mice. In the past amyloidosis has been identified as the most common non-neoplastic disease in aged CD-1 mice (Frith & Chandra 1991) but recent data from CD-1 mice shows a low incidence of systemic amyloidosis exclusively in females (Petterino et al., 2015). Fighting among group-housed male mice is associated with increased amyloidosis (Page & Glenner 1972; Sass 1996c). It has been noted that the occurrence of adrenal amyloidosis does not necessarily coincide with a high frequency of generalized amyloidsis (Dunn 1967; Sass 1996c; Yarrington 1996). Male inbred C3H mice have a low incidence while inbred female C57BL/6 have a mild age-associated incidence of amyloidosis. Their F1 hybrid, the B6C3F1 mouse, has a low spontaneous incidence of amyloidosis.

Amyloid deposits in the adrenal are initially seen in the zona fasiculata where the deposits appear within the cortical cells. As animals age the amyloid forms as linear deposits in the zona fasiculata generally sparing the zona glomerulosa and medulla. Amyloid deposits have an amorphous eosinophilic appearance in H&E-stained tissue and fibrillary appearance in electron micrographs. The amyloid deposits typically are not associated with cellular infiltrates and are Congo red-positive with bright green birefringence under polarized light

Lipogenic Pigment

(Figure 19-20)

As the name implies the origin of this pigmentation is from peroxidation of intracytoplasmic fat that becomes insoluble. Lipogenic pigment is a common age-associated change that affects most mouse strains and appears as a yellowish-brown granular to amorphous intracytoplasmic distention in cortical epithelial cells at the inner zona fasciculata (Frith 1996 Chapter; Yarrington 1996; Jayne 1963). Affected cortical epithelial cells become enlarged with lateral displacement of a pyknotic nucleus giving them the appearance of macrophages. With time, adjacent affected cells coalesce into multinucleated clusters filled with foamy light brownish to yellowish-brown pigment, sometime forming a complete band of pigmented epithelia at the cortico-medullary interface. When extensive, pigment deposition may appear in the medulla. While it is possible that the location of pigment deposition is related to the X-zone, it is noted that a similar location of lipogenic pigment occurs in rats that lack a defined x-zone and in male mice where x-zone involution is early and complete at sexual maturity.

Lipogenic pigment is a common incidental finding as mice age but its deposition is exaggerated after some xenobiotic exposures, in association with adrenal cortical atrophy, and in other conditions such as vitamin E deficiency. It has been rarely seen as a treatment effect in NTP studies but was increased in severity and incidence in the inner cortex of the adrenal in the B6C3F1 mouse two-year tricresyl phosphate study (NTP TR 433). In that study there was distention of macrophages and epitheloid cells with yellow-brown cytoplasmic deposits especially prominent in females. The early appearance and increased accumulation of pigment in that study is consistent with increased steroidogenesis. Lipogenic pigment is PAS and acid-fast positive and sudanophilic and is intralysosomal by electron microscopy. Its significance is uncertain, is similar to ‘wear-and-tear’ pigment, and is considered synonymous with ceroid and lipofuscin pigment, although there is some debate in the older literature regarding the similarity of ceroid and lipofuscin. This excess lipid accumulation reflects an ongoing pathological process (Jolly and Dalefield 1989). Lipogenic pigment should be distinguished from hemosiderin deposits seen as sequelae to hemorrhage.

Cysts

(Figures 21-22)

Although rare, epithelial-lined cortical cysts can occur in the mouse adrenal and should be distinguished from angiectasis and cystic degeneration. The cyst lining may consist of cuboidal, columnar, and even ciliated cells and the lumen may contain flocculent to homogeneous pale eosinophilic material. Depending upon the size of the cyst, there may be distortion of adjacent parenchyma. Cysts would not be expected as a consequence of treatment and are unlikely to be toxicologically significant. However, diagnosis is recommended to establish a historical control database for this change.

Inflammatory and Vascular Lesions and Mineralization

(Figure 23)

Inflammatory and vascular lesions as well as mineralization are all rare in mice (https://ntp.niehs.nih.gov/nnl/endocrine/adrenal/index.htm). When seen, adrenal gland inflammation is usually associated with systemic inflammation or peritonitis. Experimental autoimmune adrenalitis was produced by immunizing inbred SMA mice with multiple injections of adrenal extract mixed with Klebsiella O3 lipopolysaccharide in a murine model for Addison’s disease (Fujii et al., 1992). After the 8^th injection the adrenal cortex was infiltrated with polymorphonuclear leukocytes to be replaced by lymphocytes and monocytes following the 9^th injection with subsequent fibrosis. The medulla was spared. The repeated injections resulted in a delayed type hypersensitivity with production of anti-adrenocortical antibodies. Injection of normal mice with spleen cells from the immunized mice resulted in adrenalitis supporting a critical role for cell-mediated immunity.

Infection of BALB/c and C57BL mice (sex not identified) with murine cytomegalovirus causes adrenalitis characterized by diffuse infiltration of macrophages and neutrophils with increased serum corticosterone 2 days after infection (Price et al., 1996). The increased corticosterone levels reflect the previously established activation of the hypothalamic-pituitary-adrenal axis associated with acute inflammation. Both inflammatory and corticosterone responses were less prominent in C57BL mice. While the adrenal response did not mediate thymic atrophy in infected mice, the increased corticosterone levels were essential for survival up to 3 days post-infection.

Adrenal fibrosis is an uncommon finding and when present is often secondary to other processes such as infarcts and repair of prior inflammation. Adrenalitis should be differentiated from extramedullary hematopoiesis and lymphoma infiltrates.

The adrenal is highly vascularized and may have prominent sinusoids. Angiectasis of adrenal cortical sinusoids must be distinguished from hemorrhage as well as hemangioma. Angiectasis is more common in older mice and can be associated with inflammatory and degenerative changes. Distinguishing angiectasis from hemangioma is always a challenge in any tissue and depends upon the number and appearance of the endothelial component in the lesion. Adrenal hemorrhage would appear as extravasated blood and can be associated with infarction. Hemorrhage and congestion in B6C3F1 mice was associated with 2-year study exposures to 4-vinylcyclohexene (NTP TR303) and pentachloroanisole (NTP TR414).

Dystrophic mineralization is typically secondary to other changes, particularly effects causing tissue necrosis, hemorrhage, or infarction. It appears as discrete aggregates of fine to coarse deposits that stain strongly basophilic.

Hematopoietic Cell Proliferation

Although rare, hematopoietic cell proliferation in the adrenal cortex as well as in other tissues may occur secondary to ulcerative dermatitis or other significant tissue inflammatory responses or metastatic disease affecting the bone marrow. A specific example immature granulocytic cells was present in the adrenal cortex secondary to urogenital tract and other tissue inflammation in B6C3F1 mice exposed to furosemide (NTP TR 356).

Degeneration, Atrophy, and Necrosis

(Figures 24-25)

3-Methylsulphonyl-DDE (MeSO2-DDE), the metabolite of DDT, is a highly tissue-specific toxicant to the fetal and postnatal adrenal zona fasciculata in C57BL mice (Jonsson et al., 1992). Pups that have suckled dams treated with MeSO@-DDE have degenerative changes as well as extensive vacuolization and necrosis of the zona fasciculata. It is speculated that the tissue-specific metabolism to the active metabolite of DDT in the fetal and postnatal adrenal cortex is mediated by CYP 11beta.

Hernandez-Pando and co-workers (Hernandex-Pando et al., 1995) experimentally infected mice by intratracheal instillation of Mycobacterium tuberculosis. The infected mice initially developed adrenal cortical hyperplasia that morphologically resembled ACTH-driven Cushing’s disease followed within 3 weeks by progressive atrophy of all cortical layers even though the adrenals were not infected. The switch to the atrophic process was associated with cortical cell apoptosis and the development of the IgG1 response to the Mycobacterium. The authors speculated that the effects were due to T-cell dysfunction and immunologically mediated tissue damage possibly associated with cytokine release affecting the HPA axis.

Extensive adrenocortical degeneration, cystic degeneration, “brown degeneration” and atropy are documented in the older literature following treatment of three inbred strains of male mice with the anticonvulsant 5,5-diphenyhydantoin (Dilantin) sodium (Staple 1954). The mice were given daily intraperitoneal injections of Dilantin for 16 weeks. There was some peritonitis documented but other endocrine tissues were histologically normal.

Aerosol exposure to mycotoxin T-2 has been shown to induce necrosis of the inner cortex in castrated males, castrated females, and intact female mice. This necrosis was prevented in all three mouse groups by prior injection of 12 rounds of testosterone propionate at 48-hour intervals (Thurman et al., 1989). Lymphocytolysis occurred in the thumus of all mice confirming the presence of systemic mycotoxicosis.

Premature & Delayed X-zone Involution

(Figures 26-28)

Premature X-zone involution has been seen in several instances in NTP B6C3F1 subacute and chronic toxicity studies. Female mice at the age of 18 to 20 weeks may have fatty vacuolization present in the x-zone as the normal age-associate involution of this zone, although fatty vacuolization is not a necessary component of x-zone involution. In 18-20 week-old female mice exposed to acetonitrile, fatty vacuolization was absent in the x-zone, suggesting stress- or exposure-related accelerated x-zone involution (NTR TR447, 1996). In contrast to the controls, B6C3F1 female mice treated with tetrahydrofuran had a thinner than normal X-zone without the expected presence of adipocytes (NTP TR 475, 1997). There was also uterine atrophy in these mice. It was not determined if the adrenal effect was due to premature aging, a hormonal response, or a direct effect of the tetrahydrofuran. In other subchronic 90-day NTP studies in B6C3F1 mice treated with o-nitrotoluene, 1-bromopropane and androstenedione premature x-zone involution was seen in treated females versus controls. The andromimetic effect of androstenedione is a reasonable explanation for premature x-zone involution and the degree of involution was dose-dependent. There is not a clear explanation for accelerated x-zone involution in the o-nitrotoluene and 1-bromopropane exposed females.

Just as there can be premature involution of the x-zone, delayed or persistence of the x-zone may occur as a consequence of altered hormonal influence such as following gonadectomy or as a phenotype in genetically modified mice (http://www.informatics.jax.org/mp/annotations/MP:0008297). However, x-zone persistence is not commonly documented in conventional mice.

Hyperplastic, Hypertrophic and Neoplastic Lesions – Adrenal Cortex

Cortical hyperplastic and hypertrophic lesions and medullary hyperplastic lesions are age-related changes in mice. Consistent with conventional definitions, hyperplasias consist of localized increases in cell numbers. Based on site of origin, there are two diagnostic categories of cortical hyperplasia and neoplasia, subcapsular and zona fasiculata. The subcapsular hyperplasias can progress to benign and malignant neoplasms while the cortical hyperplasias in the zona fasciculata infrequently progress to neoplasms. Medullary hyperplasias and neoplasias arise and are confined to the medulla when small but can extend to the cortex and beyond when malignant.

Subcapsular Hyperplasia

(Figures 29-31)

Subcapsular hyperplasia is very common in both male and female mice, seen as early as 4 months of age, may be unilateral or bilateral, and increases in incidence with age (Yarrington 1996; Jayne 1963). It has been reported in 59% of male and 91% of female ICR mice at age 19 months (Yoshida et al., 1986). In the NTP historical control database covering the period 2004 to 2016, the incidence of capsular/subcapsular hyperplasia ranged from 74 to 100% with no predominant sex difference. The diagnosis of capsular hyperplasia versus subcapsular hyperplasia is simply a reflection of the nomenclature preference of the study pathologist.

The earliest subcapsular lesion is a fusiform type A cell seen beneath the capsule but not affecting the zona glomerulosa. This proliferative change progresses by both vertical and horizon expansion with the addition of polygonal type B cells as the proliferative response grows (Dunn 1970). Lipid droplets are present in the cytoplasm of type A and type B cells. Ultrastructural features of the early proliferating subcapsular fusiform cells have basement membranes, desmosomes, and dense bodies that develop into intracytoplasmic vacuoles (Yoshida et al., 1986; Kim et al., 2005). The subcapsular proliferative response may be focal, multifocal, diffuse, or sometimes involve the entire capsule (Goodman 1996). Large foci of hyperplasia may become spherical and have a glandular appearance and extension into the zona fasciculata may occur.

The fusiform type A cells have flat to ovoid chromatin-rich nuclei and very scant cytoplasm that is distinctly basophilic. Polygonal type B cells have abundant cytoplasm that may contain lipid vacuoles. Mitoses are rare. Subcapsular hyperplasias may distort the normal shape of the adrenal and cause minimal compression of cortical parenchyma.

The actual cells of origin of subcapsular hyperplasia are possibly blastemic or reserve zona glomerulosa cells (Kim et al., 2005). The pathogenesis of subcapsular hyperplasias may be related to hormonally induced changes in steroidogenesis in sexually mature mice and is more common in aging female mice than in male mice (Yoshida et al., 1986) with increases in both sexes following gonadectomy (Woolley et al., 1940; Wolley 1950; Bernichstein et al., 2008; Dorner et al., 2016). It is believed that subcapsular stem or progenitor cells give rise to the subcapsular response with proliferating cells that resemble gonadal stroma and produce sex steroids (Dorner et al., 2016). An increased incidence has been associated with stress (Chvedoff et al., 1980; Unno et al., 2016). The distinction between subcapsular hyperplasia and subcapsular adenoma is based on extent of lesion and degree of compression or amount of extension above the adrenal capsule.

Subcapsular Adenoma

(Figures 32-33)

Subcapsular adenoma are usually composed of spindeloid cells, are 6 mm or greater in diameter and seen primarily in old mice. Tumor cells are arranged in cords and packets with more evidence of cellular proliferation in comparison to hyperplasia. If the adenoma contains polygonal cells they are often larger than what is seen in hyperplasias. (Frith and Dunn 1994; Longeart et al., 1996). Distinction of adenomas from hyperplasia is based on prominent compression of the zona fasiculata and sometimes compression of the medulla. The normal architecture of the adrenal gland may be distorted or destroyed.

Subcapsular adenomas morphologically appear to progressive arise from hyperplasia although this is not obligatory (Dunn 1970). Adenomas comprised of polygonal cells appear to be hormonally active with estrogenic, androgenic and even adrenocorticoid activity (Dunn 1970; Frantz and Kirschbaum 1949). Subcapsular adenomas in castrated male mice may secrete androgens.

Subcapsular Carcinoma

(Figure 34)

Subcapsular carcinomas are larger and more invasive than adenoma and may metastasize with primary metastasis to the lung (Longeart et al., 1996). Carcinomas are composed of spindeloid cells arranged in sheets and may have a herringbone pattern. Cellular pleomorphism and mitotic figures are common in carcinomas (Frith and Dunn 1994). Extension beyond the capsule is a useful diagnostic criterion for malignancy.

Cortical Hyperplasia

Well demarcated, small, and usually rounded collections of small cells that tinctorially differ from the zona fasiculata may occur in the adrenal cortex. There is some debate regarding whether these are true hyperplastic lesions or functionally altered clusters of cells that are smaller than adjacent zona fasiculata epithelia. These lesions are generally diagnosed as focal hyperplasias because the increased cellular density implies a proliferative response. Larger well circumscribed hyperplasia may occasionally be present in mice without compression or distortion of the adrenal cortex parenchyma. Focal lesions consisting of both hyperplastic and hypertrophic cells can also occur and are diagnosed based on the most prevalent feature with a description of the mixed microscopic feature provided in the pathology narrative. Adrenal cortical hyperplasia resembling adrenocorticotrophic-driven changes resembling Cushing’s disease are initial responses seen in male BALB/c mice with pulmonary tuberculosis (Hernandez-Pando et al., 1995). Disruption of the pituitary-adrenal-gonadal endocrine axis with increased ACTH secretion follow castration can induce cortical hyperplasia (Strickland et al., 1980).

Cortical Adenoma

(Figures 35-36)

While benign and malignant tumors arising within the cortical zona fasciculata may occur as distinct from subcapsular neoplasms (see above), these are not common (Heath 1996). There is a morphological continuum from cortical hyperplasia to adenoma. Diagnosis of adenoma is based primarily on compression of adjacent parenchymal tissues. There is some confusion in the literature where cortical adenoma has been lumped with subcapsular adenoma. The cytological and architectural features of cortical adenoma and subcapsular adenoma are sufficiently distinctive and should be kept separate (Capen et al., 2001).

Hyperplastic, Hypertrophic and Neoplastic Lesions – Adrenal medulla

While non-neoplastic adrenal medullary lesions such as necrosis, infarction, mineralization, and hemorrhage are certainly plausible events, published documented examples are lacking. Changes commonly seen at the corticomedullary junction such as pigmentation and x-zone changes may extend into the medulla but are typically classified as cortical changes.

Medulla Hyperplasia

(Figures 37-38)

Adrenal medullary hyperplasia is common enough that at least a few occurrences can be seen in most chronic mouse studies. The NTP historical control range during the period 2004 to 2016 is 0% to 20% with no differences between male and female B6C3F1 mice. Focal as well as diffuse medullary hyperplasia in chronic CD-1 mouse studies has been reported as only up to approximately 1% in both sexes (Petterino et al., 2015). Medullary hyperplasia is often focal but occasionally may be diffuse and is frequently identified because of increased basophilia of the hyperplastic cells. The hyperplasic cells may be smaller or larger than normal chromaffin cells without compression of adjacent parenchyma or intrusion into the cortex. Medullar hyperplasia varies from very small focal aggregates to up to almost 50% of the normal medullary size. Larger hyperplasias may be associated with an increased width of the medulla and some intrusion into the cortex (Longeart et al., 1996). Mouse chromaffin cells proliferate throughout lifetime and the capacity to proliferate is believed to be strain-dependent. Chromaffin cell proliferative capacity varies among species with rats>mice> human>bovine (Tischler 1996). There is an apparent morphological continuum between medullary hyperplasia and pheochromocytoma (Greaves 2012; Longeart 1996; Chandra et al., 2013).

Pheochromocytoma

(Figures 39-40)

Age-related spontaneous pheochromocytomas are rare and typically unilateral in different mouse strains (Tischler & Sheldon 1996; Petterino et al., 2015; Haseman et al., 1998; Tischler et al., 1996; Longeart 1996; Frith & Dunn 1994). Pheochromocytomas vary in size and may be quite small. Large pheochromocytomas macroscopically appear as dark nodularity to the affected adrenal. Histopathologic features include growth in sheets and small clusters that sometimes form trabecular patterns. A hallmark of almost all pheochromocytomas is a polymorphous growth pattern with cell size ranging from small to large and with prominent cellular pleomorphism. Cytologic features can vary from spindeloid cells to cells that resemble neurons. Distension of capillaries, hemorrhage and necrosis is often present (Tischler et al., 1996; Longeart 1996; Chandra et al., 2013; Greaves 2012). Mitotic activity is highly variable and cortical infiltration may be difficult to assess particularly with involvement of hilar structures (Tischler and Sheldon 1996). For more differentiated pheochromocytomas the primary diagnostic distinction from chromaffin hyperplasia is clearly evident compression of adjacent more normal tissues. The distinction between benign and malignant pheochromocytomas is based on penetration of the adrenal capsule and/or adjacent soft tissue, invasion of blood vessels, or metastasis (Tischler & Sheldon 1996; Capen et al., 2001).

The highly variable phenotype of mouse phenochromocytomas is quite comparable to human pheochromocytomas and, unlike rat pheochromocytomas, mouse pheochromocytomas are more polymorphous and tend to be unilateral (Gorgas and Boc 1976; Longeart 1996; Tischler & Sheldon 1996; Tischler et al., 1996). The immunohistochemical expression of phenylethanolamine-N-methytransferase (PNMT) is common to mouse and human but not to rat pheochromocytomas (Hill et al., 2003). Studies on mouse pheochromocytoma immunohistochemisty have documented positive but variable responses to chromogranin A (CGA), tyrosine hydroxylase (TH), and PNMT in medullary chromaffin cells (Hill et al., 2003; Tischler and Sheldon 1996). PNMT is the rate-limiting step in conversion of norepinephrine to epinephrine and is, therefore, specific for epinephrine secreting chromaffin cells while CGA and TH immunopositivity is present in both norepinephrine and epinephrine containing chromaffin cells. These findings indicate that pheochromocytomas are functionally similar to normal adrenal medullary catecholamine synthesis.

Compared to the rat, the low incidence of spontaneous pheochromocytoma and a reduced susceptibility of chemical induction of pheochromocytomas in mice is postulated to be associated with lower susceptility to chromaffin cell proliferation in the mouse (Tischler et al., 1997; Chandra et al., 2013). Treatment-related increased incidences of pheochromocytomas in male B6C3F1 mice were documented in NTP studies of 4-hexylresorcinol (NTP TR330), 4,4-methylenedianiline HCl (NTP TR248 ), and technical grade pentachlorophenol (NTP TR349) and in both sexes for Dowicide EC-7 phentchlorophenol (NTP TR349) and furan (NTP TR 402).

Complex Pheochromocytomas

The origin of pheochromocytomas from sympathoblasts provides an opportunity for development of pheochromocytomas comprised of a mixture of chromaffin cells, neuroblasts, ganglion cells, Schwann cells and neurofibrils (Longeart et al., 1996; Capen et al., 2001). This sympathoblast origin largely explains the remarkable pleomorphism of pheochromocytomas in mice. A diagnosis of complex pheochromocytoma is applied when the neural component is present but at less than 80%. Otherwise a diagnosis of neuroblastoma might be more appropriate. Ganglion cells that may be present are typically large, polyhedral and have abundant cytoplasm (Longeart et al., 1996).

Ganglioneuroma

Originating from neural crest sympathogonia, ganglioneuroma can theoretically occur in mice and would be diagnosed when consisting of well differentiated ganglion cells with smaller components of nerve fibers and supporting neural cells.

Neuroblastoma

(Figure 41)

Neuroblastomas are comprised primarily of neuroblasts (>80%) and have a tightly compact growth pattern with occasional rosette formation (Longeart et al., 1996). Ultrastructurally, the presence of a network of axonic processes and a core of membrane-bound vesicles has been documented (Tischler and Sheldon 1996).

Adrenal Effects of Stress

Most studies related to understanding the effects of stress have been done in rat models but are presumably applicable to mouse studies. Acute stress is generally seen in study durations under one week. Since stress reponses involve the HPA axis as well as the sympathetic-adrenomedullary axis, acute and chronic stress responses can manifest in the adrenal cortex as well as the medulla.

General indicators of stress in experimental and toxicity studies include alterations in subsets of lymphocytes, changes in circulating leukocytes, increase in circulating corticosteroids, and increases in adrenal weight, with adrenal weight being a better indicator of stress than circulating corticosteroids (Everds et al., 2013). The initial cortical response to stress is hypertrophy of zona fasciculata cells. If stress is prolonged, hyperplasia is seen in the zona fasciculata and zone glomerulosa. It should be kept in mind that by just relying on microscopy, it is not possible to differentiate adrenocortical hypertrophy secondary to stress from hypertrophy as a pharmaco-toxicological perturbation of one or more steps in the steroidogenesis cascade (Harvey & Sutcliffe 2010). In both cases the hypertrophy is a response to increased ACTH secretion with increase glucocorticoids in the stress response but inhibition of glucocorticoid synthesis as a response to direct toxicity. Up to 60 chemicals with different steroidogenic targets have been identified as examples of non-stress hypertrophy adrenocortical responses (Harvey et al., 2007). Hypertrophy as a stress response will typically be associated with a stress leukogram (lymphopenia, eosinopenia, mature neutrophilia), lymphocytolysis and thymic atrophy.

There have been two approaches to studying stress in rodents: effects of housing/caging conditions/population density and use of specific stress manipulations. Rodent stress manipulation models include immobilization, cold exposure, foot shock, isolation, forced exercise, noise, and subordinate housing. With respect to housing and population density studies, there is some inconsistency but in general increased density favors mouse strain-dependent eustress and reduced or no change in adrenal weight (Paigen et al., 2016). Voluntary exercise also promotes eustress. On the other hand, forced exercise leads to acute stress with activation of the HPA and renin-angiotensin systems, increase ACTH secretion, increased plasma glucocorticoids, and increased adrenal weight. BALB/c mice stressed by restraint for 1 day to 3 weeks have increased circulating ACTH and glucocorticoids, increased adrenal weight, and decreased cholesterol content reflecting activation of the HPA axis (Hayashi et al., 2014). Animal age is important in evaluating adrenal weight increase since normal adrenal weight in mice increases from 3 to 7 weeks of age and then stabilizes in females but undergoes a 25% reduction between 7 and 9 weeks in males (Bielohuby et al., 2007). In chronic stress there is an increased resetting of basal corticosterone levels, decrease in neutral lipid vacuolation, increase in adrenocortical cell mitochondria and increased cortical cell hyperplasia (Everds et al., 2013). Cytoplasmic vacuolation associated with stress should be more diffuse and bilateral in contrast to background focal vacuolation change that is typically unilateral. Subordinate housing (confinement with a dominant male) of 19 days duration in CD-1 mice as a chronic stressor was associated with increased adrenal weight, decreased adrenocortical cell responsiveness to ACTH, and lower plasma corticosteroid to ACTH ratio (Reber et al., 2007; Fuchsi et al., 2014).

Adrenal medullary effects of acute stress include degranulation of chromaffin cells and increased secretion of epinephrine and norepinephrine. The change is associated with decreased reuptake and metabolism of catecholamines. An increase in PNMT mRNA in the medulla occurs secondary to promotion of transcription by increased cortical glucocorticoids, reflection the important interaction between cortex and medulla. Medullary effects of chronic stress include increased catecholamine synthesis, increase proliferation of chromaffin cells, and an increased frequency of pheochromocytomas (Rosol et al., 2001; Everds et al., 2013) and are dependent on the duration and repetition of stress. In a mouse model of depression, after 7 days of unpredictable stress in male C57BL/6 mice, there was no depressive-like behavior but there was increased mRNA catecholamine synthesis and increased capacity of chromaffin cells to store and release catecholamines (Santana et al., 2015). Following 21 days of unpredictable chronic stress there was decreased catecholamine synthesis and impairment of adrenal medullary function and development of depressive-like behavior. Using a model of chronic psycho-social stress, male C57BL/6 mice exposed to subordinate colony housing for 19 days had reduced body weight gain, thymic atrophy, adrenal hypertrophy, and increased norepinephrine secretion that was directly or indirectly involved in development of colitis (Reber et al., 2007). In a model of restraint-induced stress both adrenocortical and adrenomedullary effects in male BALB/c mice were seen (Hayashi et al., 2014). After 1 to 3 weeks there was increase ACTH and glucorticoid secretion, increase adrenal weight, and decreased adrenal cholesterol that returned to control levels after 4 weeks of stress. In the same mice there was increased chromogranin A that is co-secreted with catecholamines from 1 day to 2 weeks with a subsequent decrease after 3 and 4 weeks of stress. Both adrenocortical and adrenomedullary responses reflect changes influenced by the duration and frequency of stress.

Immunohistochemistry

Adrenal Cortex

While publications using immunohistochemistry for human adrenal glands are abundant, far fewer papers on mouse adrenocortical immunohistochemistry are found and most deal with signaling that accompanies development during gestation. In a study of differentiation and proliferative markers during adrenal gland development, BrdU positive cells co-localized with the differentiation marker 3-beta-hydroxysteroid dehydrogenase and additionally these two immunohistochemical markers co-localized with in situ hyrbridization demonstration of the ACTH receptor MC2-R. Condensation of all three markers appears in the subcapsular compartment during late gestation and early post-natal life (Schulte et al., 2007). Transient renin immunopositivity may be related to adrenal morphogenesis since it has been documented in fetal and neonatal adrenal cortical cells, although it is no longer present 3 days after birth (Kon et al., 1990). Using in situ hybridization and immunohistochemistry, Kiiveri and coworkers show that GATA-4 is likely important only during fetal adrenal development while GATA-6 is needed throughout adrenal development from fetal to adult age (Kiiveri et al., 2002). While TGFbeta1 expression has been demonstrated in the zona fasciculata of adult mice (Thompson et al., 1989), immunohistochemisty of developing mouse embryos show strong expression of TGFbeta1 and TGFbeta2 starting as early as gestation day 15 (Pelton et al., 1991).

Routine immunohistochemistry for PCNA, BrdU and Ki67 for detection of cell proliferation in the adult rodent adrenal cortex is routine (Chang et al., 2013) and special staining for intracytoplasmic fat that would be associated with steroidogenesis should be straightforward. However, it should be noted that localization of steroidogenesis in specific adrenocortical cells may not distinguish between sites of synthesis versus sites of storage. In an ICH comparison to localize androgen receptor in different mouse, rat, and human tissues, tissue localization is similar in all three species with immunopositivity clearly present in the adrenal zona fasiculata cells of adult CD-1 mice (Takeda et al., 1990). Using the gonadectomized model of adrenal cortical neoplasia, LH receptor expression based on immunohistochemistry is present in cortex of ovariectomized DBA/2J, C57BL/6J, and F1 hybrids but not in intact mice (Bernichtein et al., 2008).

Adrenal Medulla

Special staining of adrenomedullary cells and lesions has been reported for identification of sites of epinephrine, nonepinephrine, chromogranins A, cynaptophysin and neuron-specifc enolase using readily available commercial antibodies. Staining for tyrosine hydroxylase, the rate-limiting enzyme in catecholamine synthesis, is highy specific (Tischler et al., 1996). Other enzyme markers that can be used for identification of chromaffin cells are dopamine b-hydroxylase and phenylethanolamine-N-methytransferase. These latter two markers identify cellular localization for production of nonepinephrine from dopamine and epinephrine from norepinephrine, respectively. A comparative immunohistochemical study of spontaneous and chemically induced pheochromocytomas in B6C3F1 mice assessed the expression of phenylethanolamine-N-methyltransferase (PMNT), tyrosine hydrozylase (TH), and chrommagranin A (CGA) in spontaneous and chemically induced pheochrommocytomas (Hill et al., 2003). The study objective was to characterize functional features of mouse pheochromocytomas. All 14 spontaneous and 27 treatment-induced pheochromocytomas were strongly positive for tyrosine hydroxylase indicating that these tumors would most likely be able to synthesize catecholamines. A highly significant difference between benign and malignant pheochromocytomas was identified using PMNT immunostaining with all spontaneous and 3 of four chemical treatments that produced benign pheochromocytomas showing immunopositivity while all malignant pheochromocytomas were negative for PMNT immunoreactivity.  PMNT is apparently highly regulated in chromaffin cells with loss of reactivity in pheochromocytomas with aggressive characteristics. CGA immunostaining was variable with immunopositivity for spontaneous benign and malignant pheochromocytomas along with immunopositivity for 3 of the 4 chemical treatments that produced pheochromocytomas, reflecting the neuroendocrine features of the pheochromocytomas. Gamma-aminobutyric acid (GABA) immunoreactivity has been demonstrated in mouse adrenal chromaffin cells by light and electron microscopy with GABA-positive chromaffin cells co-expressing noradrenalin (Oomori et al., 1993).

Genetically Engineered Mouse and Cell Culture Models

Adrenal Cortex

Mouse models of adrenal cortical tumorigenesis include gonadectomy-induced adrenal cortical tumors in conventional mice, models involving serial passage of transplantable cortical tumors, transgenic models involving expression of oncogenes, mouse models with targeted genetic deletions, and mouse models with defective adrenal growth and development (Hantel and Beuschlein, 2010). Gonadectomy-induced adrenocortical neoplasia has been identified in rats, guinea pigs, hamsters and ferrets in addition to the well-known multiple strain mouse models (Woolley et al., 1940; Woolley 1950; Bernichstein et al., 2008; Dorner et al., 2016). Details related to signaling molecules and pathways implicated in gonadectomy-induced adrenal cortical neoplasia involve a major causative mechanism related to chronic elevation of gonadotrophins (Bielenska et al., 2006). Gonadectomy at 1 month of age in transgenic mice carrying SV40 T-antigen under the influence of the mouse inhibin alpha-subunit develop adrenocortical neoplasia at age 6 to 8 months (Kananen et al., 1996). These cortical tumors apparently arise in the X-zone and do not develop in non-gonadectomized littermates, suggesting that gonadal inhibin downregulates expression of the inhibin alpha-subunit gene in the adrenal gland. Another transgenic mouse model carrying fetal globin/SV40 T antigen and developing adrenocortical tumors has been described as possibly a result of T-antigen expression in embryonic adrenal tissue (Perez-Stable et al., 1996).

Adrenocortical cell culture models can be used to study the molecular and biochemical mechanisms controlling steroidogenesis and defining obligatory components of ACTH responsiveness. Y-1 is the original and most common mouse cell culture model and variants include the protein kinase A-deficient Kin-8 model and several ACTH receptor-deficient clones of the Y-1 cell model (Rainey et al., 2004; Schimmer et al., 1995; Clark and Hudson, 2015). The Y-1 model originated from an inbred LAF1 mouse irradiated during Operation Greenhouse, the American atomic bomb nuclear test exercise conducted at the Pacific Proving Ground islands of Enewetak Atoll in 1951 (Hantel and Beuschlein, 2010). One LAF1 mouse exposed to an atomic blast developed a radiation-induced adrenocortical tumor in the right adrenal gland that has been maintained as a transplantable cell line and the source for the Y-1 cell lines and its variants.

Adrenal Medulla

Genetically engineered mouse models of adrenal medullary pathologies, especially pheochromocytomas and neuroblastomas, have been developed using mutant genes and viral oncogenes. Adrenal medullary tumors can be produced in transgenic mice either by targeting of viral oncogenes to catecholamine- producing tissues or by knocking out specific genetic sequences with the tumor phenotype influenced by the genetic alteration (Longeart, 1996; Tischler and Sheldon, 1996; Tischler et al., 1996). Mice with germline mutations in the murine nuerofibromatosis (NF1) gene are predisposed to develop pheochromocytomas phenotypically similar to spontaneous pheochromocytomas (Jacks et al., 1994). These mice have one wild-type and one mutant allele of the NF1 gene. Adrenal medullary tumor models have been described in conventional mice injected with polyoma virus and in mice expressing SV40 T-antigen driven by different promoters (Tischler et al., 1993). SV40- mouse medullary tumors associated with a mouse hypothalamic growth hormone-releasing promoter tend to be poorly differentiated with neuroepithelial features and pseudorosette forations as well as hematogenous and lymphatic metastasis (Giraldi et al., 1994; Rindi 1994). More typical pheochromocytoma similar to natural counterparts have been associated with c-mos, MEN2B and Pten models (Tischler and Sheldon, 1996).

Transgenic mice expressing mos protooncogenes linked to the Moloney murine sarcoma virus long terminal repeat develop a high frequency of multicentric pheochromocytomas and/or medullary thyroid neoplasms after a long latency period (Schulz et al., 1992). Mutation of the protooncogene ret is proposed to be involved in the pathogenesis of human familial multiple endocrine neoplasia (MEN) type 2A with tumor pathogenesis similar to the human MEN 2 syndrome (Mulligan et al., 1993). Mice deficient in alpha-inhibin (a tumor-suppresser gene) have been described as developing gonadal and adrenal medullary tumors with nearly 100% penetrance (Kumar et al., 1995).

Adrenal medullary in vitro cell line models are important for investigating treatment modalities for human pheochromocytoma. The mouse MPC cell line was derived from a heterozygous Nf1 knockout mouse and its MTT aggressive derivative are genetically relevant to human pheochromocytomas and are being used in preclinical drug development (Tischler and Favier, 2015). Other useful adrenal medullar cell lines, including mouse cell lines, are available (Eaton and Duplan, 2004).

Practical Notes

• To avoid handling artifacts when weighing adrenals in experimental studies, they may be fixed before weighing. Combined adrenal weight is usually recorded in toxicity studies.
• Because of their small size, mouse adrenals are typically embedded wholly (without slicing) for histological preparations. Carefully microtomy should allow obtaining adequate representation of cortex and medulla.
• Adrenals are often embedded together either alone or along with pituitary gland but usually not with other larger tissues to maximize obtaining good representation of cortex and medulla.
• Since the adrenal is a paired organ, identifying of unilateral versus bilateral may help distinguish sporadic from systemic effects. Sometimes only one adrenal is sufficiently sectioned or available for evaluation. A significant diagnosis based on the presence of only one adrenal should be noted in the pathology record, especially if the adrenal is a study target tissue.
• Age-related changes in the adrenal glands can by induced prematurely and/or exacerbated by treatment or stress to yield an increased incidence versus age-matches controls. Similarly, cortical cytological alterations can fluctuate cyclically as a normal physiological response but may also be altered by treatment. Thus, it is important to make comparison to appropriate controls to identify potential treatment-related effects.
• Severity grading may be important for some lesions to determine if treatment has exacerbated an effect. Severity grading is recommended for all hyperplastic lesions of the adrenal cortex and medulla since there is often a continuum with neoplasia. Severity grading for other changes such as lipogenic pigment deposition, necrosis, cytoplasmic vacuolization, and mineralization should also be considered if a treatment-related exacerbation is suspected.
• Because of close interplay among endocrine tissues, changes in the adrenal may be associated with effects in other endocrine organs. Important adrenal functional and morphological effects on steroidogenesis can be a direct trophic response to pituitary secretions. It is recommended that adrenal effects be evaluated along with the rest of the endocrine system.
• Premature or delayed adrenal X-zone involution is a potential treatment-related effect. Thus, comparison to appropriate age-matched and gender controls is important. Adrenal X-zone involution is uniquely susceptible to andromimetic effects.

References

Bell, C. L., and Murray, S. A. (2016). Adrenocortical gap junctions and their functions. Front Endocrinol (Lausanne) 7, 82.

Bernichtein, S., Petretto, E., Jamieson, S., Goel, A., Aitman, T. J., Mangion, J. M., and Huhtaniemi, I. T. (2008). Adrenal gland tumorigenesis after gonadectomy in mice is a complex genetic trait driven by epistatic loci. Endocrinology 149, 651-61.

Bertholet, J. Y. (1980). Proliferative activity and cell migration in the adrenal cortex of fetal and neonatal rats: An autoradiographic study. J Endocrinol 87, 1-9.

Bielinska, M., Kiiveri, S., Parviainen, H., Mannisto, S., Heikinheimo, M., and Wilson, D. B. (2006). Gonadectomy-induced adrenocortical neoplasia in the domestic ferret (mustela putorius furo) and laboratory mouse. Vet Pathol 43, 97-117.

Bielohuby, M., Herbach, N., Wanke, R., Maser-Gluth, C., Beuschlein, F., Wolf, E., and Hoeflich, A. (2007). Growth analysis of the mouse adrenal gland from weaning to adulthood: Time- and gender-dependent alterations of cell size and number in the cortical compartment. Am J Physiol Endocrinol Metab 293, E139-46.

Bozzo, A., Sonez, C., Cobeta, I., Avila, R., Rolando, A., Romanini, M., Lazarte, M., Gauna, H., and Mugnaini, M. (2011). Chronic stress effects on adrenal cortex cellular proliferation in pregrnant rats. Int J Morphol 29, 1148-1157.

Capen, C., Karbe, E., and al., e. (2001). Endocrine system. In International classification of rodent tumors (U. Mohr, ed., pp. 269-322. Springer, Berlin.

Chandra, S., Hoenerhoff, M., and Peterson, R. (2013). Endocrine glands. In Toxicologic pathology. Nonclinical safety assessment (P. Sahota, J. Popp, J. Hardisty and C. Gopinath, eds.), pp. 680-692. CRC Press, Boca Raton.

Chvedoff, M., Clarke, M. R., Irisarri, E., Faccini, J. M., and Monro, A. M. (1980). Effects of housing conditions on food intake, body weight and spontaneous lesions in mice. A review of the literature and results of an 18-month study. Food Cosmet Toxicol 18, 517-22.

Clark, B. J., and Hudson, E. A. (2015). Star protein stability in y1 and kin-8 mouse adrenocortical cells. Biology (Basel) 4, 200-15.

Daughaday, W. (1941). A comparison of the x-zone of the adrenal cortex in two inbred strains of mice. Cancer Res 1, 883-885.

Deacon, C. F., Mosley, W., and Jones, I. C. (1986). The x zone of the mouse adrenal cortex of the swiss albino strain. Gen Comp Endocrinol 61, 87-99.

Dorner, J., Martinez Rodriguez, V., Ziegler, R., Rohrig, T., Cochran, R. S., Gotz, R. M., Levin, M. D., Pihlajoki, M., Heikinheimo, M., and Wilson, D. B. (2017). Gli1+ progenitor cells in the adrenal capsule of the adult mouse give rise to heterotopic gonadal-like tissue. Mol Cell Endocrinol 441, 164-175.

Dunn TB (1967) Amyloidosis in mice. In: Pathology of Laboratory Rats and Mice. Cotchin E, Roe FJC (eds). Blackwell Scientific, Oxoford. pp 181-212.

Dunn, T. B. (1970). Normal and pathologic anatomy of the adrenal gland of the mouse, including neoplasms. J Natl Cancer Inst 44, 1323-89.

Eaton, M. J., and Duplan, H. (2004). Useful cell lines derived from the adrenal medulla. Mol Cell Endocrinol 228, 39-52.

Ehrhart-Bornstein, M., and Hilbers, U. (1998). Neuuroendocrine properties of adrenocortical cells. Horm Metab Res 30, 436-439.

Eranko, O. (1955). Histochemistry of noradrenaline in the adrenal medulla of rats and mice. Endocrinology 57, 363-8.

Everds, N. E., Snyder, P. W., Bailey, K. L., Bolon, B., Creasy, D. M., Foley, G. L., Rosol, T. J., and Sellers, T. (2013). Interpreting stress responses during routine toxicity studies: A review of the biology, impact, and assessment. Toxicol Pathol 41, 560-614.

Frantz, M., and Kirschbaum, A. (1949). Sex hormone secretion by tumors
of the adrenal cortex of mice. Cancer Res 9, 257-266.

Frith CH (1983) Histology, adrenal gland, mouse. In: Endocrine System. Jones TC, Mohr U and Hunt RD (eds). Springer-Verlag, Berlin. pp 8-12.

Frith CH and Dunn TB (1994) Tumours of the adrenal gland. In: Pathology of Tumours in Laboratory Animals. Tumours of the Mouse, II Edition. Turusov VS and Mohr U (eds). IARC Scientific Publications No. 111, Lyon, France. pp 595-609.

Frith CH (1996a) Histology, Adrenal Gland, Mouse. In: Endocrine System. Jones TC, Capen CC and Mohr U (eds). Springer-Verlag Berlin Heidelberg. pp 386-391.

Frith, C. H., and Chandra, M. (1991). Incidence, distribution, and morphology of amyloidosis in charles rivers cd-1 mice. Toxicol Pathol 19, 123-7.

Frith CH (1996b) Lipogenic pigmentation, adrenal cortex, Mouse. In:
Endocrine System.. Jones TC, Capen CC and Mohr U (eds). Springer- Verlag Berlin Heidelberg. pp 458-462.

Fuchsl, A. M., Neumann, I. D., and Reber, S. O. (2014). Stress resilience: A low-anxiety genotype protects male mice from the consequences of chronic psychosocial stress. Endocrinology 155, 117-26.

Fujii, Y., Kato, N., Kito, J., Asai, J., and Yokochi, T. (1992). Experimental autoimmune adrenalitis: A murine model for addison’s disease. Autoimmunity 12, 47-52.

Gersh, I., and Grollman, A. (1939). The nature of the x-zone of the adrenal gland of the mouse. The Anatomical Record 75, 131-153.

Goodman DG (1996) Subcapsular-cell hyperplasia, adrenal, Mouse. In: Endocrine System. Jones TC, Capen CC and Mohr U (eds). Springer-Verlag Berlin, Heidelberg. pp 464-467.

Gorgas, K., and Bock, P. (1976). Morphology and histochemistry of the adrenal medulla. I. Various types of primary catecholamine-storing cells in the mouse adrenal medulla. Histochemistry 50, 17-31.

Greaves, P. (2012). Histopathology of preclinical toxicity studies. Elsevier, Amsterdam. 886 pages.

Hantel, C., and Beuschlein, F. (2010). Mouse models of adrenal tumorigenesis. Best Pract Res Clin Endocrinol Metab 24, 865-75.

Harvey, P. W., Everett, D. J., and Springall, C. J. (2007). Adrenal toxicology: A strategy for assessment of functional toxicity to the adrenal cortex and steroidogenesis. J Appl Toxicol 27, 103-15.

Harvey, P. W., and Sutcliffe, C. (2010). Adrenocortical hypertrophy: Establishing cause and toxicological significance. J Appl Toxicol 30, 617-26.

Haseman, J. K., Hailey, J. R., and Morris, R. W. (1998). Spontaneous neoplasm incidences in fischer 344 rats and b6c3f1 mice in two-year carcinogenicity studies: A national toxicology program update. Toxicol Pathol 26, 428-41.

Hayashi, T., Ikematsu, K., Abe, Y., Ihama, Y., Ago, K., Ago, M., Miyazaki, T., and Ogata, M. (2014). Temporal changes of the adrenal endocrine system in a restraint stressed mouse and possibility of postmortem indicators of prolonged psychological stress. Leg Med (Tokyo) 16, 193-6.

Hernandez-Pando, R., Orozco, H., Honour, J., Silva, P., Leyva, R., and Rook, G. A. (1995). Adrenal changes in murine pulmonary tuberculosis; a clue to pathogenesis? FEMS Immunol Med Microbiol 12, 63-72.

Hill, G. D., Pace, V., Persohn, E., Bresser, C., Haseman, J. K., Tischler, A. S., and Nyska, A. (2003). A comparative immunohistochemical study of spontaneous and chemically induced pheochromocytomas in b6c3f1 mice. Endocr Pathol 14, 81-91.

Hoeflich, A., and Bielohuby, M. (2009). Mechanisms of adrenal gland growth: Signal integration by extracellular signal regulated kinases1/2. J Mol Endocrinol 42, 191-203.

Howard-Miller, E. (1927). A transitory zone in the adrenal cortex which shows age and sex relationships. Am J Anat 40, 251-293.

Huang, C. C., Liu, C., and Yao, H. H. (2012). Investigating the role of adrenal cortex in organization and differentiation of the adrenal medulla in mice. Mol Cell Endocrinol 361, 165-71.

Huang, C. C., Miyagawa, S., Matsumaru, D., Parker, K. L., and Yao, H. H. (2010). Progenitor cell expansion and organ size of mouse adrenal is regulated by sonic hedgehog. Endocrinology 151, 1119-28.

Hummel, K. P. (1958). Accessory adrenal cortical nodules in the mouse. Anat Rec 132, 281-95.

Jacks, T., Shih, T. S., Schmitt, E. M., Bronson, R. T., Bernards, A., and Weinberg, R. A. (1994). Tumour predisposition in mice heterozygous for a targeted mutation in nf1. Nat Genet 7, 353-61.

Jayne, E. P. (1963). A histo-cytologic study of the adrenal cortex in mice as influenced by strain, sex, and age. J Gerontol 18, 227-34.

Jolly RD and Dalefield RR (1989) Lipopigments in veterinary pathology: Pathogenesis and terminology. Adv Exp Med Biol 266:157-168.

Jones, I. C. (1948). Variation in the mouse adrenal cortex with special reference to the zona reticularis and to brown degeneration, together with a discussion of the cell migration theory. Q J Microsc Sci 89, 53-74.

Jones, I. C. (1952). The disappearance of the x zone of the mouse adrenal cortex during first pregnancy. Proc R Soc Lond B Biol Sci 139, 398-410.

Jonsson, C. J., Lund, B. O., Bergman, A., and Brandt, I. (1992). Adrenocortical toxicity of 3-methylsulphonyl-dde; 3: Studies in fetal and suckling mice. Reprod Toxicol 6, 233-40.

Kananen, K., Markkula, M., Mikola, M., Rainio, E. M., McNeilly, A., and Huhtaniemi, I. (1996). Gonadectomy permits adrenocortical tumorigenesis in mice transgenic for the mouse inhibin alpha-subunit promoter/simian virus 40 t-antigen fusion gene: Evidence for negative autoregulation of the inhibin alpha-subunit gene. Mol Endocrinol 10, 1667-77.

Kataoka, Y., Ikehara, Y., and Hattori, T. (1996). Cell proliferation and renewal of mouse adrenal cortex. J Anat 188 ( Pt 2), 375-81.

Kaufman, M., Nikitin, A., and Sundberg, J. (2010). Histologic Basis of Mouse Endocrine System Development. CRC Press, Boca Raton. 240 pages.

Kiiveri, S., Liu, J., Westerholm-Ormio, M., Narita, N., Wilson, D. B., Voutilainen, R., and Heikinheimo, M. (2002). Differential expression of gata-4 and gata-6 in fetal and adult mouse and human adrenal tissue. Endocrinology 143, 3136-43.

Kim, J. S., Kubota, H., Sakai, T., Doi, K., and Saegusa, J. (2005). Electron microscopic study of subcapsular cell hyperplasia in the adrenal glands of iqi/jic mice. Exp Anim 54, 107-10.

Kon, Y., Hashimoto, Y., Kitagawa, H., Sugimura, M., and Murakami, K. (1990). Renin immunohistochemistry in the adrenal gland of the mouse fetus and neonate. Anat Rec 227, 124-31.

Kumar TR, Donehower LA, Bradley A and Matzuk MM (1995) Transgenic mouse models for tumor-suppressor genes. J Intern Med 238:233-238.

Laufer, E., Kesper, D., Vortkamp, A., and King, P. (2012). Sonic hedgehog signaling during adrenal development. Mol Cell Endocrinol 351, 19-27.

Longeart LE (1996) Adrenal medullary tumors, mouse. In: Endocrine System.. Jones TC, Capen CC and Mohr U (eds). Springer-Verlag, Berlin. pp 421-427.

McNicol, A. M., and Duffy, A. E. (1987). A study of cell migration in the adrenal cortex of the rat using bromodeoxyuridine. Cell Tissue Kinet 20, 519-26.

McPhail, M., and Read, H. (1942). The mouse adrenal. I. Development, degeneration and regeneration of the x-zone. The Anatomical Record 84, 51-73.

Mulligan LM, Kwok JB, Healey CS, Eldson MJ, Eng C, Gardner E, Loce DR, Mole SE, Moore JK, Papi L, Ponder MA, Telenius H, Tunnacliffe A and Ponder MAJ (1993) Germ-line mutations in the ret proto-oncogene in multiple endocrine neoplasia type 2A. Nature 363:458-460.

Oomori, Y., Iuchi, H., Nakaya, K., Tanaka, H., Ishikawa, K., Satoh, Y., and Ono, K. (1993). Gamma-aminobutyric acid (gaba) immunoreactivity in the mouse adrenal gland. Histochemistry 100, 203-13.

Paigen, B., Currer, J. M., and Svenson, K. L. (2016). Effects of varied housing density on a hybrid mouse strain followed for 20 months. PLoS One 11, e0149647.

Pecori Giraldi, F., Mizobuchi, M., Horowitz, Z. D., Downs, T. R., Aleppo, G., Kier, A., Wagner, T., Yun, J. S., Kopchick, J. J., and Frohman, L. A. (1994). Development of neuroepithelial tumors of the adrenal medulla in transgenic mice expressing a mouse hypothalamic growth hormone-releasing hormone promoter-simian virus-40 t-antigen fusion gene. Endocrinology 134, 1219-24.

Pelton, R. W., Saxena, B., Jones, M., Moses, H. L., and Gold, L. I. (1991). Immunohistochemical localization of tgf beta 1, tgf beta 2, and tgf beta 3 in the mouse embryo: Expression patterns suggest multiple roles during embryonic development. J Cell Biol 115, 1091-105.

Perez-Stable, C., Altman, N. H., Brown, J., Harbison, M., Cray, C., and Roos, B. A. (1996). Prostate, adrenocortical, and brown adipose tumors in fetal globin/t antigen transgenic mice. Lab Invest 74, 363-73.

Petterino, C., Naylor, S., Mukaratirwa, S., and Bradley, A. (2015). Adrenal gland background findings in cd-1 (crl:Cd-1(icr)br) mice from 104-week carcinogenicity studies. Toxicol Pathol 43, 816-24.

Price, P., Olver, S. D., Silich, M., Nador, T. Z., Yerkovich, S., and Wilson, S. G. (1996). Adrenalitis and the adrenocortical response of resistant and susceptible mice to acute murine cytomegalovirus infection. Eur J Clin Invest 26, 811-9.

Rainey, W. E., Saner, K., and Schimmer, B. P. (2004). Adrenocortical cell lines. Mol Cell Endocrinol 228, 23-38.

Reber, S. O., Birkeneder, L., Veenema, A. H., Obermeier, F., Falk, W., Straub, R. H., and Neumann, I. D. (2007). Adrenal insufficiency and colonic inflammation after a novel chronic psycho-social stress paradigm in mice: Implications and mechanisms. Endocrinology 148, 670-82.

Rindi, G. (1994). Characterisation of neuroendocrine tumors in transgenic mice. Digestion 55 Suppl 3, 24-30.

Rosol, T. J., Yarrington, J. T., Latendresse, J., and Capen, C. C. (2001). Adrenal gland: Structure, function, and mechanisms of toxicity. Toxicol Pathol 29, 41-8.

Santana, M. M., Rosmaninho-Salgado, J., Cortez, V., Pereira, F. C., Kaster, M. P., Aveleira, C. A., Ferreira, M., Alvaro, A. R., and Cavadas, C. (2015). Impaired adrenal medullary function in a mouse model of depression induced by unpredictable chronic stress. Eur Neuropsychopharmacol 25, 1753-66.

Sass B (1996a) Embryology, adrenal gland, mouse. In: Endocrine System. Jones TC, Capen CC and Mohr U (eds). Springer-Verlag, Berlin, Heidelberg, New York. pp 381-386.

Sass B (1996b) Accessory adrenocortical tissue, mouse. In: Endocrine System. Jones TC, Capen CC and Mohr U (eds). Springer-Verlag, Berlin, Heidelberg, New York. pp 391-394.

Sass B (1996c) Amyloidosis, adrenal, mouse. In: Endocrine System. Jones TC, Capen CC and Mohr U (eds). Springer-Verlag, Berlin, Heidelberg, New York. pp 455-458.

Schimmer, B. P., Kwan, W. K., Tsao, J., and Qiu, R. (1995). Acth-receptor deficient mutants of the y1 mouse adrenocortical tumor cell line. Endocr Res 21, 139-56.

Schulte, D. M., Shapiro, I., Reincke, M., and Beuschlein, F. (2007). Expression and spatio-temporal distribution of differentiation and proliferation markers during mouse adrenal development. Gene Expr Patterns 7, 72-81.

Schulz N, Propst F, Rosenberg MP, Linnoila RI, Paules RS, Kovatch R, Ogiso Y and Vande-Woude G (1992) Pheochromocytomas and C-cell thyroid neoplasms in transgenic c-mos mice: a model for the human multiple endocrine neoplasia type 2 syndrome. Cancer Res 52:450-455.

Staple, P. H. (1954). The effects of continued administration of 5:5-diphenythydantoin (dilantin) sodium on the adrenal glands in mice. J R Microsc Soc 74, 10-21.

Starkey, W., and Schmidt, C. (1938). The effect of testosterone-propionate on the x-zone of the mouse adrenal. Endocrinol 23, 339-344.

Strickland, J. E., Saviolakis, G. A., Weislow, O. S., Allen, P. T., Hellman, A., and Fowler, A. K. (1980). Spontaneous adrenal tumors in the aged, ovariectomized nih swiss mouse without enhanced retrovirus expression. Cancer Res 40, 3570-5.

Sucheston, M., and Cannon, M. (1972). The transient-zone in the human and mouse adrenal gland. The Ohio Journal of Science 72, 120-126.

Suto, J. (2012). Quantitative trait locus mapping of genes associated with vacuolation in the adrenal x-zone of the ddd/sgn inbred mouse. BMC Genet 13, 95.

Takeda, H., Chodak, G., Mutchnik, S., Nakamoto, T., and Chang, C. (1990). Immunohistochemical localization of androgen receptors with mono- and polyclonal antibodies to androgen receptor. J Endocrinol 126, 17-25.

Tanaka, S., and Matsuzawa, A. (1995). Comparison of adrenocortical zonation in c57bl/6j and ddd mice. Exp Anim 44, 285-91.

Thompson, N. L., Flanders, K. C., Smith, J. M., Ellingsworth, L. R., Roberts, A. B., and Sporn, M. B. (1989). Expression of transforming growth factor-beta 1 in specific cells and tissues of adult and neonatal mice. J Cell Biol 108, 661-9.

Thurman, J. D., Creasia, D. A., and Trotter, R. W. (1989). Effects of testosterone on the prevention of t-2 toxin-induced adrenocortical necrosis in mice. Am J Vet Res 50, 942-4.

Tischler, A., and Favier, J. (2015). Models of pheochromocytoma: What’s on the horizon? Int J Endo Oncol 2, 171-174.

Tischler, A. S., Freund, R., Carroll, J., Cahill, A. L., Perlman, R. L., Alroy, J., and Riseberg, J. C. (1993). Polyoma-induced neoplasms of the mouse adrenal medulla. Characterization of the tumors and establishment of cell lines. Lab Invest 68, 541-9.

Tischler, A. S., Powers, J. F., Shahsavari, M., Ziar, J., Tsokas, P., Downing, J., and McClain, R. M. (1997). Comparative studies of chromaffin cell proliferation in the adrenal medulla of rats and mice. Fundam Appl Toxicol 35, 216-20.

Tischler, A. S., Sheldon, W., and Gray, R. (1996). Immunohistochemical and morphological characterization of spontaneously occurring pheochromocytomas in the aging mouse. Vet Pathol 33, 512-20.

Tischler AS, Freund R, Carroll J, Cahill A, Perlman RL, Alroy J and Riseberg J (1993) Polyoma-induced neoplasms of the mouse adrenal medulla. Characterization of the tumors and establishment of cell lines. Lab Invest 68:541-549.

Tischler AS and Sheldon W (1996) Adrenal medulla. In: Pathobiology of the Aging Mouse. Mohr U, Dungworth DL, Capen CC, Carlton WW, Sundberg JP and Ward J (eds). ILSI Press Washington D.C. pp 135-151.

Tsujio, M., Mizorogi, T., Nishijima, K., Kuwahara, S., Aoyama, H., Ohno, T., and Tanaka, S. (2009). A morphometric study of the adrenal cortex of the female ddd mouse. J Vet Med Sci 71, 183-7.

Ungar, F., and Stabler, T. A. (1980). 20 alpha-hydroxysteroid dehydrogenase activity and the x-zone of the female mouse adrenal. J Steroid Biochem 13, 23-8.

Unno, K., Hara, A., Nakagawa, A., Iguchi, K., Ohshio, M., Morita, A., and Nakamura, Y. (2016). Anti-stress effects of drinking green tea with lowered caffeine and enriched theanine, epigallocatechin and arginine on psychosocial stress induced adrenal hypertrophy in mice. Phytomedicine 23, 1365-1374.

Walczak, E. M., and Hammer, G. D. (2015). Regulation of the adrenocortical stem cell niche: Implications for disease. Nat Rev Endocrinol 11, 14-28.

Waring, H., and Scott, E. (1937). Some abnormalities of the adrenal gland of the mouse with a discussion on cortical homology. J Anat 71, 299-321.

Woolley GW (1950) Experimental endocrine tumours with special reference to the adrenal cortex. Rec Progr Horm Res 5:383-405.

Woolley, G., Fekete, E., and Little, C. (1940). Effect of castration in the dilute brown strain of mice. Endocrinology 28, 341-343.

Wright, N. A., Voncina, D., and Morley, A. R. (1973). An attempt to demonstrate cell migration from the zona glomerulosa in the prepubertal male rat adrenal cortex. J Endocrinol 59, 451-9.

Yarrington JT (1996) Adrenal cortex. In: Pathobiology of the Aging Mouse. Mohr U, Dungworth DL, Capen CC, Carlton WW, Sundberg JP and Ward JM (eds). ILSI Press Washington D.C. pp 125-133.

Yoshida, A., Maita, K., and Shirasu, Y. (1986). Subcapsular cell hyperplasia in the mouse adrenal glands. Nihon Juigaku Zasshi 48, 719-28.

Zajicek, G., Ariel, I., and Arber, N. (1986). The streaming adrenal cortex: Direct evidence of centripetal migration of adrenocytes by estimation of cell turnover rate. J Endocrinol 111, 477-82.