INHAND: Non-proliferative and proliferative lesions of fish – Chapter 4. Fish endocrine system

Introduction

From a physiological perspective, there is a high degree of functional similarity between piscine and mammalian endocrine systems. Accordingly, hormonal signaling molecules, hormone receptors, and stimulatory and inhibitory feedback loops are all surprisingly well-conserved among these distant vertebrate taxa. Conversely, the structural composition and anatomic distribution of endocrine tissues vary markedly between mammals and fish, and to a somewhat lesser degree among fish species; examples of this variability will be highlighted in the discussion to follow. Histopathologic evidence of spontaneous lesions or induced effects involving fish endocrine glands is relatively uncommon in toxicological studies, aside from a few notable exceptions in selected species (e.g., ultimobranchial gland tumors in zebrafish Danio rerio).

Major endocrine tissues in fish include: chromaffin and interrenal cells, the corpuscles of Stannius, islets of the endocrine pancreas, pineal gland, pituitary gland, thyroid follicles, ultimobranchial gland, and the urophysis. Excluded from this category, but discussed elsewhere in this issue, are additional tissues that are capable of producing endocrine or paracrine signals, such as the gastrointestinal tract, gonads, heart, kidney, leukocytes, and liver.

Necropsy and Trimming

Because fish endocrine organs are small and often buried within other tissues, they are rarely excised specifically for routine examination in toxicological studies, and this is true even for investigations that involve larger fish species. Exceptions include: interrenal and chromaffin tissues, which may appear in histologic sections if certain areas of anterior kidney are sampled; the corpuscles of Stannius, which may be collected intentionally or unintentionally with segments of posterior kidney; islets of endocrine pancreas, which may be present in areas of mesenteric pancreas collected with gut specimens; and the pituitary gland which may remain attached to the brain if it is sampled. Additionally, special effort can be made to excise certain tissues, e.g., pharyngeal tissue that contains thyroid follicles, if they are suspected to be toxicologic targets. More commonly, endocrine organs are visualized in whole-body sections of small fish species, or in sections of larvae or juveniles of large fishes. However, due to their diminutive size, endocrine tissues can be difficult to recover consistently in whole-body sections, and thus the acquisition of multiple step sections at and near the sagittal midline (where many endocrine organs are located) may be required.

Morphology, Physiology, Nomenclature, Diagnostic Criteria, and Differential Diagnosis

1. Chromaffin and interrenal cells

(Figs. 4.1, 4.2, 4.3, and 4.4)

Fig. 4.1.

Bluegill, juvenile. Anterior Kidney. Normal. Chromaffin and interrenal tissue surrounds a branch of the cardinal vein in the anterior kidney. Note the additional presence of a few urinary tubules. Parasagittal section. H&E. Image courtesy of Jeffrey Wolf.

Fig. 4.2.

Bluegill. Anterior kidney. Normal. Chromaffin and interrenal tissue (higher magnification of Fig. 4.1). The relatively fewer chromaffin cells have paler cytoplasm than the interrenal cells. H&E. Image courtesy of Jeffrey Wolf.

Fig. 4.3.

Guppy, female. Anterior kidney. Normal. Chromaffin and interrenal tissue. Parasagittal section. H&E. Image courtesy of Jeffrey Wolf.

Fig. 4.4.

Zebrafish. Anterior kidney. Anisocytosis and anisokaryosis in otherwise normal chromaffin and interrenal cells. H&E.

Table 1 shows the nomenclature, diagnostic criteria, and differential diagnoses pertaining to the chromaffin and interrenal cells.

Table 1. Nomenclature, diagnostic criteria, and differential diagnoses pertaining to the chromaffin and interrenal cells.

Chromaffin and interrenal cells		Common	Uncommon	Not observed, but potentially relevant	Not applicable
Non-proliferative
	*Anisocytosis/anisokaryosis		X
	*Atrophy		X
	*Hypertrophy		X
	Necrosis		X
Proliferative
	Non-neoplastic
	*Hyperplasia		X

*Terminology with diagnostic criteria or comments described in the main text.

Chromaffin and interrenal cells are the piscine analogues of adrenal medullary and cortical tissue, respectively. Unlike the encapsulated adrenal glands of mammals, in teleost fish these tissues are typically located as linear or patchy cords of cells in the anterior kidney, often immediately adjacent to or near the posterior cardinal veins. The interrenal and chromaffin cells are intermingled in some species and separated in others¹, and if there is a difference between the cell types that can be appreciated in H&E sections, it is that interrenal cell cytoplasm typically stains more intensely pink compared to the paler chromaffin cells.

Chromaffin cells produce catecholamines that include epinephrine, norepinephrine, and lesser amounts of dopamine. In addition to serving as a component of the fish stress response, catecholemines have roles in regulating gill blood flow and oxygen uptake. The major hormone produced by interrenal cells is cortisol, followed by lesser amounts of corticosterone, deoxycorticosterone, and aldosterone.

Interestingly, cortisol also functions as a mineralocorticoid in fish.

Chromaffin and Interrenal Cells – Non-Proliferative

Comments

Spontaneous and induced lesions involving the interrenal or chromaffin cells are uncommon. Because of their location within the anterior kidney they may be inadevertently affected by renal inflammation or other kidney diseases.

Anisocytosis/anisokaryosis, chromaffin or interrenal cells

Other Terms

• Cellular pleomorphism

• Megalocytosis

Pathogenesis

• A non-uniform increase or reduction of cellular or nuclear material, in the absence of cytokinesis, among cells of similar origin/type. This manifests as variation in cell or nuclear size, including ploidy (karyomegaly and/or anisokaryosis)

Diagnostic Features

• Variation in cell cytoplasmic and nuclear size

Differential Diagnoses

• Autolysis

Comments

This has been observed anecdotally as an incidental finding in zebrafish. There is no evidence as yet to suspect that this can be a treatment-induced lesion. Unless accompanied by cell proliferation, with or without the formation of a mass, this finding should probably be interpreted as a benign background change.

Atrophy, chromaffin or interrenal cells

Other Terms

• Chromaffin cells, reduced; Interrenal cells, reduced

Pathogenesis

• A reduction of cellular material such that a reduction in cell size is clearly evident, possibly due to reduced stimulus

Diagnostic Features

• Chromaffin or interrenal cells are smaller than expected but typically display normal cytologic features

Differential Diagnoses

• Autolysis

Comments

Atrophy of chromaffin or interrenal tissue has not been reported as a spontaneous finding and appears to be rare as an induced effect. Interrenal cell atrophy, associated with low blood cortisol levels, was reported in northern pike Esox lucius and yellow perch Perca flavescens that were exposed to bleached kraft mill effluent².

A diagnosis of atrophy should be based on evidence of decreased cell size and not merely a decrease in the amount of tissue present, as the amount of chromaffin/interrenal tissue visualized in a single histologic section is highly variable.

Stereological methods may be useful for confirming decreases in chromaffin or interrenal cell size.

Nomenclature recommendation

• Interrenal or chromaffin cells can alternatively be referred to as interrenal or chromaffin tissue

Hypertrophy, chromaffin or interrenal cells

Other Terms

• Chromaffin cells, enlarged

• Interrenal cells, enlarged

Pathogenesis

• An increase of cellular material such that an enlargement in cell size is clearly evident, possibly due to increased stimulus

Diagnostic Features

• Chromaffin or interrenal cells are enlarged and may have enlarged nuclei

Differential Diagnoses

• Vacuolation

• Autolysis

Comments

Interrenal cell hypertrophy was reported in fish collected from an urban stream³ and from water contaminated with metals such as cadmium and zinc⁴. It has been proposed that hypertrophy of these cell types may be a response to chronic physiological stress. It is unknown whether chromaffin or interrenal cell hyperplasia is associated with increased catecholamine or corticosteroid secretion, respectively.

Chromaffin and Interrenal Cells – Proliferative Non-Neoplastic

Hyperplasia, chromaffin or interrenal cells

(Fig. 4.5)

Fig. 4.5.

Guppy, female. Anterior kidney. Hyperplasia, chromaffin cell. Parasagittal section. H&E. Image courtesy of Jeffrey Wolf.

Other Terms

• Chromaffin cells, increased

• Interrenal cells, increased

Pathogenesis

• An increase in cell numbers as a result of incread mitosis or reduced apoptosis in the tissue population

Diagnostic Features

• Chromaffin or interrenal cells may be slightly enlarged but typically display normal cytologic features

Differential Diagnoses

• Chromaffinoma / pheochromocytoma

• Granulomatous inflammation

Comments

The example of chromaffin cell hyperplasia illustrated in this paper occurred spontaneously in a single guppy Poecilia reticulata. Interrenal cell hyperplasia (and hypertrophy) was reported in brown trout Salmo trutta collected from water contaminated with metals such as cadmium and zinc⁴, and both chromaffin and interrenal cell hyperplasia were observed in paddlefish Polyodon spathula from water contaminated with chlordane and PCBs⁵.

Credible changes in cell numbers can be difficult to confirm, because the amount of chromaffin/interrenal tissue in a histologic section is highly dependent on the precise locations available for viewing, and the plane of section acquired.As such, morphometric (e.g., stereological) tools are used to quantify these tissues in serial or step sections. It is important to recognize that the perception of increased nuclear density can be a function of increased cell number, decreased cell size, or both. Additionally, it is not always easy to distinguish chromaffin from interrenal cells, especially for species where cell types are mixed, as well as in cases of suboptimal histologic staining. It is unknown whether hyperplastic proliferation of these cell types is associated with increased catecholamine or corticosteroid secretion.

Chromaffin and Interrenal Cells – Neoplastic

Neoplasms involving piscine chromaffin or interrenal tissues are not typically observed in toxicological studies.

2. Corpuscles of Stannius

The corpuscles of Stannius are found only in teleost and holostean fishes (the latter are jawless taxa comprised of the lampreys and hagfishes) (Fig. 4.6, Fig. 4.7, Fig. 4.8, Fig. 4.9, 4.10). These discrete, bilaterally paired, finely-encapsulated ovoid structures are typically embedded in the posterior kidney, ventrolaterally along the caudal aspect. They can be visualized macroscopically in some larger species. The anterior-posterior location of these glands varies according to species, and the right and left gland may be offset to some degree. The glands themselves consist of cells with basilar nuclei that are arranged in tightly folded cords or packets. The apical margins of the cells appose capillaries that are embedded within connective tissue septa. From a pathological perspective, it is imperative not to misdiagnose these normal structures as renal tubular adenomas.

Fig. 4.6.

Zebrafish, female. Posterior kidney. Corpuscle of Stannius. Normal (rectangle). Note close association between the corpuscle and the posterior kidney. Midline sagittal section. H&E. Image courtesy of Jeffrey Wolf.

Fig. 4.7.

Medaka. Caudal coelomic cavity. Corpuscle of Stannius. Normal. In medaka, this organ is located in association with the mesonephric duct in the caudal abdominal region. Sagittal section. H&E. Image courtesy of Jeffrey Wolf.

Fig. 4.8.

Medaka. Posterior kidney. Corpuscle of Stannius. Normal. (higher magnification of Fig. 4.7). A portion of mesonephric duct (lower and left) contains retrograde spermatozoa. H&E. Image courtesy of Jeffrey Wolf.

Fig. 4.9.

Zebrafish. Posterior kidney. Corpuscle of Stannius. Normal, H&E.

Fig. 4.10.

Zebrafish, male, aged. Posterior kidney. Corpuscle of Stannius. Normal. H&E.

Blood calcium levels in fish are regulated by hormones produced by the corpuscles of Stannius, the ultimobranchial body, and the pituitary gland. The corpuscles of Stannius secrete stanniocalcin, which reduces plasma calcium by decreasing its absorption via the gills and intestine. Conversely, calcitonin, which is produced by the ultimobranchial body (fish lack thyroid gland C-cells), is thought to reduce calcium via increased renal excretion and increased bone sequestration. Parathyroid hormone, and more notably in fish, parathyroid hormone-related protein (PTHrP), counteract these effects by increasing plasma calcium. Fish lack parathyroid glands, and production of PTHrP is thought to occur in the pituitary and possibly other peripheral sites⁶. Additional substances that may play a role in piscine calcium homeostasis include cortisol, isotocin, hydrogen sulfide, and insulin-like growth factor⁷.

Spontaneous and induced lesions involving the corpuscles of Stannius are rare. Because of their intimate anatomic association with the posterior kidney they may be affected inadvertently by renal inflammation or other kidney diseases.

Table 2 shows the nomenclature, diagnostic criteria, and differential diagnoses pertaining to the corpuscles of Stannius.

Table 2. Nomenclature, diagnostic criteria, and differential diagnoses pertaining to the corpuscles of Stannius.

Corpuscles of Stannius		Common	Uncommon	Not observed, but potentially relevant	Not applicable
Non-proliferative
	*Cyst(s)		X
	*Mineralization		X
Proliferative
	Neoplastic
	*Adenoma		X

*Terminology with diagnostic criteria or comments described in the main text.

Corpuscles of Stannius – Non-Proliferative

Cyst(s), corpuscles of Stannius

(Fig. 4.11)

Fig. 4.11.

Zebrafish, Aged. Posterior kidney, Cyst, corpuscle of Stannius. H&E.

Other Terms

• Cystic degeneration

Pathogenesis

• This is apparently an age-related change that has been observed anecdotally in zebrafish, for example. Affected zebrafish are almost always greater than 1.5 years of age. The precise pathogenesis of this lesion has not been reported

Diagnostic Features

• Single or multiple, fluid-filled, ovoid spaces

• The cysts appear to be lined by a flattened epithelium in most instances

Differential Diagnoses

• Digenean parasitic cyst

Comments

This rare incidental age-related change does not appear to be adverse, nor has it been associated with experimental treatment. The pathologist should be consistent in either recording them as incidental findings, or not recording them due to their commonality and suspected lack of toxicological significance.

Mineralization, corpuscles of Stannius

(Fig. 4.12)

Fig. 4.12.

Zebrafish, Posterior kidney. Mineralization, corpuscle of Stannius (corpora amylacea). Midline sagittal section. H&E.

Other Terms

• Corpora amylacea

• Mineralized deposits

Pathogenesis

• This is apparently an age-related change that has been observed anecdotally in zebrafish, for example. The precise pathogenesis of this lesion has not been reported

Diagnostic Features

• Single or multiple lamellated mineralized concretions located within expanded interstitial spaces

• Typically little if any cellular immunologic reaction to the mineral

Differential Diagnoses

• Migrating helminth parasites

Comments

Age-related mineralization can be observed as a spontaneous finding in a variety of fish tissues, most commonly involving the urinary (posterior) kidney and gonads. It is suspected that husbandry factors may play a role in the development of such lesions, e.g., mineral content of the water or feed, or water pH.

Corpuscles of Stannius – Proliferative Non-Neoplastic

Proliferative non-neoplastic lesions involving the corpuscle of Stannius are not typically observed in toxicological studies.

Corpuscles of Stannius - Proliferative Neoplastic

Adenoma, (B) corpuscles of Stannius

(Fig. 4.13)

Fig. 4.13.

Zebrafish. Posterior kidney. Adenoma, corpuscle of Stannius. Observed as an incidental finding in an adult female. This apparently rare lesion has not been previously reported; unencapsulated, compressive mass effect. Sagittal midline section. H&E.

Other Terms

• None

Pathogenesis

• The pathogenesis of this lesion is unknown. The case illustrated in this report was an apparently spontaneous occurrence in an untreated zebrafish

Diagnostic Features

• Well-circumscribed expansile growth that causes compression of the surrounding renal parenchyma

• The organ is larger and rounder than non-neoplastic counterpart

• Little cellular atypia

Differential Diagnoses

• Corpuscle of Stannius hyperplasia – not yet reported

• Corpuscle of Stannius carcinoma – not yet reported

• Inflammatory granuloma (see INHAND Fish Species introduction for granuloma comment)

Comments

The case in this report was an incidental finding with no known associated findings. Serum calcium was not measured.

3. Pancreas, endocrine

Compared to mammals, teleost fishes tend to have few islets of endocrine pancreas (islets of Langerhans), which are generally located within mesenteric islands of exocrine (acinar) pancreas in the anterior coelomic cavity. Typically there are one or more large primary islets called “Brockman bodies” (Figs. 4.14, 4.15, 4.16, 4.17), and up to several additional secondary islets (Fig. 4.18). The islets are discrete, finely encapsulated nodular organs that are comprised of sheets or tightly folded cords of pale cells. In certain fish species, exocrine pancreas tissue is present normally within the liver, located typically along portal tracts; conversely, endocrine islets are not generally present in the liver.

Fig. 4.14.

Zebrafish. Pancreas. Primary endocrine pancreatic islet (Brockman body). Normal. The islet is surrounded by exocrine pancreas and located within the mesentery dorsal to the liver in this case, and is associated with biliary and/or pancreatic ducts. Midline sagittal section. H&E.

Fig. 4.15.

Medaka. Pancreas. Primary endocrine pancreatic islet (Brockman body). Normal. Midline sagittal section. H&E.

Fig. 4.16.

Bluegill. Pancreas. Primary endocrine pancreatic islet (Brockman body). Normal. Midline sagittal section. H&E.

Fig. 4.17.

Channel catfish. Pancreas. Endocrine islet mixed with exocrine pancreas. Although not entirely typical for channel catfish, this occasional presentation is not necessarily considered pathologic. H&E

Fig. 4.18.

Medaka. Pancreas. Normal secondary (accessory) endocrine pancreatic islet. Midline sagittal section. H&E.

Piscine islets consist of alpha, beta and delta cells; the proportions and distributions vary depending on the species^{8, 9}. As in mammals, the alpha cells produce glucagon-like peptide which promotes hyperglycemia and gluconeogenesis, the beta cells produce insulin which promotes hypoglycemia and transport of glucose into tissues, and delta cells are the source of somatostatin which inhibits the release of insulin and glucagon. Studies have demonstrated that amino acids may actually have a greater capacity than glucose to stimulate insulin and glucagon-like peptide release¹⁰.

Endocrine pancreas (islets) may be affected as bystanders in peritoneal inflammatory diseases or intraperitoneal vaccine reactions that affect the abdominal mesentery and exocrine pancreas. In some cases, the endocrine pancreas is relatively spared, as in alphavirus infections (“pancreas disease”) of Atlantic salmon Salmo salar. There is little evidence to suggest that the endocrine pancreas is a common target of toxicants. The few published reports to date include: vacuolation and degranulation of beta cells in Clarias batrachus as a response to glibenclamide (an anti-diabetic therapeutic agent) exposure¹¹; similar effects in the alpha cells of Channa punctatus following cobalt salts exposure¹²; islet cell hyperplasia in a cutthroat trout Salmo clarki exposed to the insecticide endrin¹³; and disrupted islet organogenesis in zebrafish Danio rerio embryos exposed to perfluorooctanesulfonic acid¹⁴. Initial observations of islet cell hypertrophy or atypia, or islet gland hypertrophy, should be interpreted cautiously; such changes may be evident in untreated controls of some species, such as Japanese medaka Oryzias latipes.

Table 3 indicates nomenclature, diagnostic criteria, and differential diagnoses pertaining to the pancreas.

Table 3. Nomenclature, diagnostic criteria, and differential diagnoses pertaining to the pancreas.

Pancreas, endocrine		Common	Uncommon	Not observed, but potentially relevant	Not applicable
Non-proliferative
	Amyloid, islet			X
	*Anisocytosis / anisokaryosis, islet cell		X
	Angiectasis, islet			X
	Apoptosis, islet cell		X
	Atrophy, islet			X
	Degranulation, ß-cell			X
	Fibrosis, islet		X
	Hemorrhage, islet		X
	Hypertrophy, islet cell	X
	Infiltrate, inflammatory cell	X
	Inflammation	X
	Metaplasia, hepatocyte			X
	Necrosis, single cell		X
	Pigment, islet			X
	*Vacuolation, islet cell		X
Proliferative
	Non-neoplastic
	*Hyperplasia, islet cell		X
	Neoplastic
	Adenoma, acinar-islet cell			X
	*Adenoma, islet cell		X
	Carcinoma, acinar-islet cell			X
	*Carcinoma, islet cell		X

*Terminology with diagnostic criteria or comments described in the main text.

Endocrine Pancreas - Congenital Lesions

Congenital lesions involving the endocrine pancreas are not typically observed in toxicological studies.

Endocrine Pancreas - Non-Proliferative

Anisocytosis/anisokaryosis, islet cells, pancreas

(Figs. 4.19 and 4.20)

Fig. 4.19.

Guppy. Pancreas. Anisocytosis and anisokaryosis, primary endocrine islet. Parasagittal section. H&E. Image courtesy of Jeffrey Wolf.

Fig. 4.20.

Guppy. Pancreas. Anisocytosis and anisokaryosis, primary endocrine islet. (higher magnification of Fig. 4.19). H&E. Image courtesy of Jeffrey Wolf.

Other Terms

• Pleomorphism

• Megalocytosis

Pathogenesis

A non-uniform increase or reduction of cellular or nuclear material, in the absence of cytokinesis, among cells of similar origin/type. This manifests as variation in cell or nuclear size, including ploidy (karyomegaly and/or anisokaryosis)

Diagnostic Features

• Islet cells and islet cell nuclei are variably sized and shaped

• Surprisingly high degree of pleomorphism can be present

• Affected islets are usually well-circumscribed and of expected size for the species, age, and sex

Differential Diagnoses

• Islet cell adenoma

• Islet cell carcinoma

Comments

Observed as an incidental finding in adult guppies Poecilia reticulata, Japanese medaka Oryzias latipes, and presumably other fishes.

It is important to recognize that although fish may have large primary islets (Brockman bodies) comprised of pleomorphic cells, such islets are not necessarily neoplastic or even considered “abnormal”. Note that anisocytosis/anisokaryosis may be recorded as a combined term.

Vacuolation, islet cells, pancreas

(Figs. 4.21 and 4.22)

Fig. 4.21.

Zebrafish, kras transgenic. Pancreas. Vacuolation, endocrine islet cells. Parasagittal section. H&E.

Fig. 4.22.

Zebrafish, kras transgenic. Pancreas. Vacuolation, endocrine islet cells. Parasagittal section. H&E.

Other Terms

• Vacuolar degeneration

• Ballooning degeneration

Pathogenesis

This unusual finding was observed in transgenic zebrafish that expressed mutant kras targeted to liver (J. Spitsbergen, personal communication). Similar vacuolation of pancreatic islets occurred in carcinogenesis studies in which zebrafish were exposed to polycyclic aromatic hydrocarbons including 9,10 dimethylbenz[a]anthracene and dibenzo[a,l]pyrene¹⁵.

Diagnostic Features

• Enlarged islet cells with vacuolated adipocyte-like cytoplasm

• Multifocal to diffuse distribution

Differential Diagnoses

• Autolysis

• Granuloma formation (see INHAND Fish Species introduction)

Comments

Vacuolated islets in the pancreas of fish have been reported¹⁵.

Endocrine Pancreas - Proliferative Non-Neoplastic

Hyperplasia, islet cells, pancreas

(Fig. 4.23)

Fig. 4.23.

Zebrafish. Pancreas. Hyperplasia, extra-islet endocrine cell (nesidioblastosis); irregular proliferation of islet cells that may form duct structures with karyomegaly and cytomegaly. H&E.

Other Terms

• Increased pancreatic islets

• Islet cell proliferation

• Nesidioblastosis (extra-islet)

Pathogenesis

• An increase in cell numbers as a result of increased mitosis or reduced apoptosis in the tissue population

Diagnostic Features

• Markedly enlarged islets

• Growth pattern similar to that of normal islets

• Little if any cytological atypia

• Distinct border between endocrine and exocrine pancreas

Differential Diagnoses

• Islet cell adenoma

• Islet cell carcinoma

Comments

Islet cell hyperplasia has not generally been observed as a spontaneous finding, although there has been some association made between islet mass and spawning in salmonids¹⁶. One of the most convincing reports of islet cell hyperplasia involved cutthroat trout Salmo clarki, in which greater than one third of male and female fish developed this condition following year-long experimental exposure to the insecticide endrin¹³. Both extra-islet proliferation or hyperplasia of islets cells may be observed as spontaneous or induced findings. Observations and the literature also support a role for metaplastic transformation of duct cells to budding islet endocrine cells, reported as nesidioblastosis¹⁷. Given the variation in presentation and definition, the descriptive terms of hyperplasia or metaplasia are recommended rather than nesidioblastosis.

Many fish species have large primary islets (Brockman bodies), and it is imperative not to misdiagnose these normal anatomic structures as hyperplastic or neoplastic islets.

Endocrine Pancreas - Proliferative Neoplastic

Adenoma (B), islet cells, pancreas

(Figs. 4.24 and 4.25)

Fig. 4.24.

Zebrafish. Pancreas. Adenoma, endocrine cell. Parasagittal section. H&E.

Fig. 4.25.

Zebrafish. Pancreas. Adenoma, endocrine cell with a pseudorosette pattern (higher magnification of Fig. 4.24). H&E.

Other Terms

• Benign islet cell tumor

• Benign insulinoma

Pathogenesis/Cell of Origin

• Islet cell tumors have been observed spontaneously in wild caught hagfish Myxine glutinosa¹⁸

Diagnostic Features

• Enlarged islet

• Cells may form rosette and/or pseudorosette patterns

• Expansile lesion, capsular integrity maintained

Differential Diagnoses

• Islet cell anisocytosis / anisokaryosis – no compression, variable cell size

• Islet cell hyperplasia – increased number of cells but no capsule or compression/expansion of cells

• Islet cell carcinoma – local invasion and lack of capsule integrity

Comments

Islet cell tumors are observed occasionally in various fish species, but the literature contains only rare reports.

Carcinoma, (M) islet cells, pancreas

(Figs. 4.26, 4.27, and 4.28)

Fig. 4.26.

Medaka. Pancreas. Carcinoma, islet cell. Parasagittal section. H&E.

Fig. 4.27.

Zebrafish. Pancreas. Carcinoma, islet cell. Parasagittal section. H&E.

Fig. 4.28.

Zebrafish. Pancreas. Carcinoma, islet cell with a pseudorosette pattern. Parasagittal section. H&E.

Other Terms

• Malignant islet cell tumor

• Malignant insulinoma

Pathogenesis

Islet cell carcinomas have been created specifically via targeted expression of human MYCN in transgenic zebrafish¹⁹.

Diagnostic Features

• Enlarged islet

• Cells may form rosette and/or pseudorosette patterns

• The key diagnostic feature is compromise of the islet capsular membrane and local tissue invasion.

Differential Diagnoses

• Islet cell anisocytosis / anisokaryosis

• Islet cell hyperplasia

• Islet cell adenoma

Comments

Islet cell tumors are observed occasionally in various fish species, but the literature contains only rare reports.

4. Pineal gland (organ)

The pineal organ is located adjacent to the skull at the thinnest area of the dorsal cranial bone and it is connected to the brain via a stalk that arises from the diencephalon at the habenular nuclei (at the junction between the telencephalon and mesencephalon). The lumina of the pineal gland stalk and associated saccus dorsalis (all components of the epithalamus) connect to the lumen of the third ventricle (Figs. 4.29, 4.30 and 4.31). The pineal organ can be visualized best in sections that are midline sagittal to slightly left parasagittal, and the structures may not be evident unless the ideal section has been captured.

Fig. 4.29.

Zebrafish. Pineal gland. Normal. A stalk (incomplete in this image) connects the dorsal skull to the base of the pineal gland, which is located caudal to the telencephalon and rostral to the mesencephalon (large box region). Pineal gland outlined by the small box. Midline sagittal section. H&E. Image courtesy of Jeffrey Wolf.

Fig. 4.30.

Zebrafish. Pineal gland. Pineal epiphysis. H&E. Image courtesy of Jeffrey Wolf.

Fig. 4.31.

Trout. Juvenile Pineal gland. Normal. Midline sagittal section. H&E.

The pineal organ has both neurosensory and secretory components. The pinealocytes are photoreceptor cells that detect light transmitted through a translucent region of the dorsal skull. The principal hormone secreted by the pineal gland is melatonin, which is an indole amine secreted mainly during the dark phase of day. Melatonin synchronizes physiological and behavioral activities with photoperiod and temporal factors (i.e., time of day and season), and as such affects growth, reproduction, migration, metabolic rate, the immune system, and skin pigmentation²⁰.

Spontaneous or induced lesions of the piscine pineal organ are not typically observed in toxicological studies.

Table 4 indicates nomenclature, diagnostic criteria, and differential diagnoses pertaining to the pineal gland.

Table 4. Nomenclature, diagnostic criteria, and differential diagnoses pertaining to the pineal gland.

Pineal gland		Common	Uncommon	Not observed, but potentially relevant	Not applicable
Non-proliferative
	Fibrosis			X
	Infiltrate, inflammatory cell		X
	Inflammation		X
	Mineralization			X
	Striated muscle fibers			X
	Vacuolation			X
Proliferative
	Neoplastic
	Pinealoma, benign			X
	Pinealoma, malignant			X

5. Pituitary

Located along the axial midline, the piscine pituitary is a plump or elongated ovoid structure which in most laboratory teleosts is suspended ventral to the diencephalon by a short narrow infundibular stalk (leptobasic type) (Figs. 4.32, 4.33 and 4.34). The pituitary gland may be partially encased in a bony sella turcica (e.g., zebrafish) or not surrounded at all by bone (e.g., Japanese medaka Oryzias latipes). As in mammals, the primary components of the pituitary are the adenohypophysis and the neurohypophysis, which interdigitate extensively. The adenohypophysis consists of the pars distalis and the posteriorly located pars intermedia. Based on morphologic and functional characteristics, the pars distalis can be further divided into rostral and caudal portions. Located in the rostral pars distalis are the eosinophilic-staining prolactin cells, adrenocorticotrophic hormone (ACTH)-secreting cells, and the non-granular chromophobe cells. The caudal pars distalis contains somatotropic, gonadotropic, and thyrotropic cells. Cells that produce melanocyte stimulating hormone (MSH) are the chief component of the pars intermedia. The vascularized pars nervosa is comprised of neurosecretory fibers and glial pituicytes. The precise arrangement and proportional representation of the various hypophyseal subdivisions differ markedly among fish species. For example, in zebrafish there is a sizeable rostral extension of the neurohypophysis that is designated as the anterior pars nervosa²¹. There is no hypothalamic-hypophyseal portal vascular system in most teleosts; instead, the pituitary receives input from the hypothalamus via neurons.

Fig. 4.32.

Medaka. Pituitary, normal. Pituitary is located beneath the diencephalon and caudal to the optic nerves. The pars distalis and caudal pars nervosa are evident in this image. The bright eosinophilic cells in the ventral pituitary are prolactin secreting. Midline sagittal section. H&E. Image courtesy of Jeffrey Wolf.

Fig. 4.33.

Trout. Pituitary. Normal. This organ is dorsoventrally flattened in trout. Midline sagittal section. H&E.

Fig. 4.34.

Zebrafish. Pituitary, normal. Unlike the medaka pituitary, the zebrafish pituitary is surrounded by bone and features both an anterior and posterior pars nervosa. Midline sagittal section. H&E. Image courtesy of Jeffrey Wolf.

As in mammals, the piscine pituitary produces a variety of hormones that modulate functions in a number of other endocrine glands and organ systems. These include adrenocorticotropic hormone, gonadotropic hormones (e.g., follicle stimulating and luteinizing hormones), growth hormone, prolactin, somatolactin, and thyroid stimulating hormone. In most instances, the function of these pituitary messengers is analogous to their mammalian counterparts, but exceptions exist. For example, rather than stimulation of milk production and release, the predominant role of prolactin in fish is osmoregulatory²².

Although many studies have demonstrated putative disruption of pituitary function in fishes as a result of experimental hormonal perturbation, there are few reliable accounts of toxicologically-induced morphologic changes in the piscine pituitary. For example, Kirubagaran and Joy report that degeneration, degranulation, hypertrophy, and hyperplasia of pituitary ACTH-producing cells occurred following exposure to mercuric chloride; however, based on the included figure photomicrographs, those findings have little credibility due to substandard quality of the histologic preparation²³. Additionally, caution should be exercised when comparing the numbers and proportions of various adenohypophyseal cell types among animals and treatment groups, because cellular components can vary markedly depending on the precise plane of histologic sectioning in each animal. Furthermore, there is evidence that the relative sizes and numbers of different hormone-secreting cells may differ according to developmental stage, age, endogenous and exogenous hormonal signals, and reproductive status in fish that spawn seasonally^24,25,26.

Table 5 indicates nomenclature, diagnostic criteria, and differential diagnoses partaining to the pituitary.

Table 5. Nomenclature, diagnostic criteria, and differential diagnoses partaining to the pituitary.

Pituitary		Common	Uncommon	Not observed, but potentially relevant	Not applicable
Congenital
	Aberrant craniopharyngeal structures				X
	Aplasia/hypoplasia			X
	Cysts			X
	Persistence, Rathke’s pouch				X
Non-proliferative
	Angiectasis		X
	Atrophy			X
	*Degeneration		X
	Fibrosis			X
	Hemorrhage		X
	Hypertrophy, pars distalis			X
	Hypertrophy, pars intermedia			X
	Infiltrate, inflammatory cell		X
	Inflammation		X
	Osseous metaplasia			X
	Pigment			X
	Pseudocyst			X
	Thrombus			X
	*Vacuolation		X
Proliferative
	Non-neoplastic
	Hyperplasia, pars distalis			X
	Hyperplasia, pars intermedia			X
	Neoplastic
	*Adenoma, pars distalis		X
	Adenoma, pars intermedia			X
	Carcinoma, pars distalis			X
	Carcinoma, pars intermedia			X
	Craniopharyngioma, benign			X
	Craniopharyngioma, malignant			X
	Ganglioneuroma, benign			X
	Pituicytoma, benign		X
	Pituicytoma, malignant			X

*Terminology with diagnostic criteria or comments described in the main text.

Pituitary - Congenital Lesions

Congenital lesions of the piscine pituitary are not typically observed in toxicological studies, but cysts or pseudocysts might be observed.

Pituitary - Non-Proliferative

Degeneration, cystic, pituitary

Other terms

Pseudocyst

Pathogenesis

• Cystic spaces may develop as a consequence of genetic variations, congenital defects, or subsequent to degeneration or necrosis; the mechanism is poorly understood

Diagnostic Features

• Variably-sized cystic spaces that often contain colloid-like fluid

• Usually single, but may be multilocular, depending on the species

• Lined by parenchymal cells or a limiting membrane rather than by epithelial cells

Differential Diagnoses

• Pituitary/hypophyseal cyst – lined by goblet cells or epithelial cells

• Necrosis

Comments

Cystic degeneration has been reported to occur in the prolactin zone in the rostral pars distalis of both wild-caught and captive nine-spined stickleback Pungitius pungitius²⁷. In captive specimens, cystic degeneration appeared to be related to certain husbandry factors.

Cystic degeneration and/or cyst development was observed in the adenohypophysis of platyfish Xiphophorus maculatus²⁸, in which their origin was thought to have a genetic basis, and anecdotally as an age-related change in zebrafish.

In terms of toxicologic effects, cystic degeneration of the pituitary (“pseudocysts”) were reported to occur in sheepshead minnows Cyprinodon variegatus exposed to the herbicide triluralin for 19 months²⁹, in addition to pituitary congestion and hemorrhage.

There are no reports of functional impairment associated with the presence of pituitary cysts.

Vacuolation, pituicytes– Similar to rodents

(Fig. 4.35)

Fig. 4.35.

Zebrafish. Pituitary. Vacuolation, pituicytes. H&E.

Pituitary - Proliferative Non-Neoplastic

Proliferative non-neoplastic lesions of the piscine pituitary are not typically observed in toxicological studies.

Pituitary - Proliferative Neoplastic

Most proliferative lesions of the fish pituitary are diagnosed as adenomas (see Pituitary, adenoma).

Adenoma, (B) pars distalis, pituitary

(Figs. 4.36 and 4.37)

Fig. 4.36.

Zebrafish. Pituitary. Adenoma. H&E

Fig. 4.37.

Zebrafish. Pituitary. Adenoma (higher magnification of Fig. 4.36). H&E.

Other Terms

• Benign pituitary tumor

Pathogenesis

Pituitary adenomas involving the adenohypophysis have been reported infrequently as rare spontaneous findings in various fish species, and such tumors usually consist of proliferating gonadotrophic cells³⁰

Diagnostic Features

• Expansion of the pars distalis due to increased number of densely-packed gonadotrophs

• Gonadotroph cells may be enlarged compared to non-neoplastic cohorts

• Compression is variable and may be difficult to discern as pituitary location may be within bone in some species (zebrafish) or surrounded by fat in many species

Differential Diagnoses

• Pituitary hyperplasia – lack of cellular pleomorphism

Comments

Diagnostically, the distinction between pituitary adenoma and hyperplasia is not entirely clear. Most of the rarely reported proliferative pituitary lesions are diagnosed as adenomas. Pituitary adenomas may be associated with the presence of gonadal tumors³¹. Transgenic zebrafish models of pituitary tumor formation have also been developed^{32, 33}.

6. Thyroid

Most teleosts do not have discrete encapsulated paired thyroid glands; instead there are loosely-arranged thyroid follicles located bilaterally near the midline, along the ventral aorta in the lower jaw (Figs. 4.38, 4.39, 4.40, and 4.41). In healthy adult fishes, the follicles are typically lined by simple squamous to cuboidal epithelium, and they are filled with homogeneous colloid material that is pink in routine H&E-stained sections.

Fig. 4.38.

Zebrafish. Thyroid, normal. The thyroid is present along the path of the ventral aorta. Midline sagittal section. H&E.

Fig. 4.39.

Zebrafish. Thyroid, normal (higher magnification of Fig. 4.38). H&E.

Fig. 4.40.

Medaka, female. Thyroid. Normal thyroid follicles. Note the lateral location caudal to the branchial chamber in medaka. The follicles of females are lined by flattened to cuboidal epithelium. Parasagittal section. 100 ×, H&E.

Fig. 4.41.

Medaka, male. Thyroid. Normal thyroid follicles. Compared to adult females, adult male medaka generally have greater numbers of follicles, and their follicles are lined by cuboidal to columnar epithelium, which can appear quite hyperplastic, even in control males. Parasagittal section. H&E. Image courtesy of Jeffrey Wolf.

Anatomically, the thyroids of Japanese medaka differ from those of other laboratory species in a couple of aspects. First, although follicles are present in the lower jaw, the bulk of the thyroid tissue is located caudal to the branchial chamber. Second, thyroid follicles in male medaka are often comparatively more abundant than those of females and other teleosts, and the epithelium is frequently columnar and proliferative-appearing, with compressed follicular lumina (Figs. 4.40 and 4.41); it is possible that this gender dichotomy may represent a hormonally-governed secondary sex characteristic, although further research would be required to confirm that speculation. In a number of fish species, ectopic thyroid follicles can regularly or occasionally be found in a variety of near-midline sites, such as the ocular choroid plexus or kidney; such ectopic tissue should not be confused with tumor metastasis. Unlike mammals, the thyroid tissue of teleosts is not associated with parafollicular c-cells; instead, the ultimobranchial body bears responsibility for Calcitonin secretion.

Physiological aspects involving the thyroid follicles and hypothalamic-pituitary-thyroid axis are highly conserved among vertebrates and fish are no exception. Thyroid stimulating hormone (TSH) secreted by the pituitary drives synthesis of L-thyroxine (T₄) in the thyroid follicle, and peripheral conversion of T₄ to the active metabolite triiodothyronine (T₃) occurs via enzymatic monodeiodination. Thyroid hormone (TH) exerts effects by binding to nuclear receptors that function as DNA-binding transcription factors, and hormone inactivation in the kidney, brain, liver, and muscle is mediated by iodothyronine- deiodinases. Thyroid hormones have roles in growth, metabolism, development, reproduction, pigment deposition, and are thought to be instrumental for the metamorphic transformation of larval to juvenile stages³⁴.

Aside from involvement in systemic or regional inflammation, it is rare to observe non-proliferative lesions, whether spontaneous or treatment-induced, in the thyroid tissue of teleosts used in toxicological studies. To some extent, that may be from failure to consistently include the lower jaw as a tissue designated for examination. Proliferative changes affecting the thyroid are relatively more common, however, and include both hyperplastic and neoplastic lesions. The frequent finding of follicular tumors within areas of follicular hyperplasia suggests that progression of hyperplasia to benign or malignant neoplasia occurs along a continuum³⁵. By necessity, criteria used to determine malignancy differ from those used for mammals. In mammals the predominant indicators of malignancy are local invasion and distant metastasis. However, fish have cells with the potential to form thyroid tissue distributed along multiple midline areas throughout their bodies, either in the form of overt follicles or embryonic cellular rests. Thus, when pituitary production of TSH increases (e.g., following exposure to a goitrogenic substance or as a result of low iodine levels), both the pharyngeal and ectopic thyroid tissue may become hyperplastic or even neoplastic. Consequently, hyperplasia of ectopic thyroid glands, with or without evidence of hyperplasia in primary thyroid locations, must be differentiated from the extremely rare metastasis of thyroid adenocarcinoma; the latter can be distinguished by aberrant growth pattern (lack of follicles lined by single epithelial layer) and/or the presence of cellular atypia.

Table 6 indicates nomenclature, diagnostic criteria, and differential diagnoses pertaining to the thyroid.

Table 6. Nomenclature, diagnostic criteria, and differential diagnoses partaining to the thyroid.

Thyroid		Common	Uncommon	Not observed, but potentially relevant	Not applicable
Congenital
	Ectopic tissue, thymus			X
	*Ectopic tissue, thyroid	X
	Persistent thyroglossal duct				X
	Ultimobranchial cyst				X
Non-proliferative
	Amyloid			X
	Atrophy			X
	Colloid alteration		X
	Cystic follicle		X
	*Degeneration, follicular cell		X
	Follicular dilatation, diffuse		X
	*Hypertrophy, follicular cell		X
	Hypertrophy, follicular cell, ectopic		X
	Infiltrate, inflammatory cell		X
	Inflammation		X
	Mineralization		X
	Pigment			X
	Thyroid dysplasia			X
Proliferative
	Non-neoplastic
	Hyperplasia, C-cell				X
	*Hyperplasia, follicular cell		X
	Hyperplasia, follicular cell, ectopic		X
	Neoplastic
	Adenoma, C-cell				X
	*Adenoma, follicular cell		X
	Adenoma, follicular cell, ectopic		X
	Carcinoma, C-cell				X
	*Carcinoma, follicular cell		X
	Carcinoma, follicular cell, ectopic		X

*Terminology with diagnostic criteria or comments described in the main text.

Thyroid - Congenital Lesions

Ectopic tissue, thyroid

(Figs. 4.42, 4.47, and 4.48)

Fig. 4.42.

Medaka. Posterior kidney. Ectopic thyroid follicular cells. Parasagittal section. H&E. Image courtesy of Jeffrey Wolf.

Fig. 4.47.

Medaka, Thyroid. Hyperplasia, follicular cell in ectopic sites. Hyperplastic follicles are evident in the eye, abdominal mesentery, liver, and spleen (those last two organs protrude ventrally from the incised carcass). H&E. Image courtesy of Jeffrey Wolf.

Fig. 4.48.

Medaka. Thyroid. Hyperplasia, follicular cell; ectopic in the vascular choroid rete of the eye. H&E. Image courtesy of Jeffrey Wolf.

Other Terms

• Ectopic thyroid follicles

Pathogenesis

Can be a normal finding in healthy fish, and is observed routinely in certain locations in particular fish species (e.g., kidneys of goldfish)

Diagnostic Features

• Typically characterized by low numbers of follicles in non-pharyngeal locations

• Well-differentiated colloid-filled follicles, lined by flattened to low cuboidal epithelium

Differential Diagnoses

• Ectopic follicular cell hyperplasia

• Cyst formation

Comments

Simple ectopic follicles can be differentiated from ectopic follicular cell hyperplasia by the amount of thyroid tissue present, which tends to be exuberant in the case of the latter. The presence of colloid could be confirmed by staining for T₃ and T₄ immunohistochemically³⁶, although this is seldom necessary due to the innocuous nature of the lesions and limited list of differential diagnoses.

Thyroid - Non-Proliferative

Degeneration, follicular cell, thyroid

(Figs. 4.43 and 4.44)

Fig. 4.43.

Medaka, male. Thyroid. Degeneration, follicular cell. Thyroid is otherwise normal (albeit hyperplastic looking). Parasagittal section. H&E. Image courtesy of Jeffrey Wolf.

Fig. 4.44.

Zebrafish, aged. Thyroid. Degeneration, follicular cell, vacuolar and hyaline; in ectopic thyroid tissue located adjacent to and embedded in the cardiac ventricle. H&E.

Other Terms

• Retained intracellular colloid

• Hyaline inclusions

Pathogenesis

• This previously unpublished lesion has been observed periodically as a spontaneous finding of low frequency in the thyroid glands of adult Japanese medaka used as negative controls in toxicologic experiments. The prevalence of this appears to be higher in males than females. The hyaline material that distends the cytoplasm of follicular epithelial cells may be inspissated colloid that, for unknown reasons, is retained intracellularly rather than secreted into the follicular lumen

Diagnostic Features

• Distension of the follicular cell cytoplasm by small to large, pale pink to glassy red, ovoid to irregular inclusions

• Larger inclusions cause peripheralization and condensation of the follicular epithelial cell nucleus

• May affect a single follicle or multiple follicles (but typically not all follicles), and some or all cells may be affected within a given follicle

Differential Diagnoses

• Viral inclusions (however, no currently known virus produces similar inclusions in the thyroid)

• Follicular cell necrosis – presence of cellular debris

Comments

There is no indication as yet that the presence of this lesion has a negative impact on affected fish or a significant impact on the outcome of toxicologic studies. It is possible that hyaline degeneration will also be observed in the thyroid follicles of other fish species, as this organ continues to be more routinely examined. A somewhat similar change was observed in ectopic thyroid tissue located adjacent to the cardiac ventricle of aged zebrafish. This unusual lesion was associated with cardiac enlargement and severe endocardiosis of the bulbo-ventricular valve³⁷.

Hypertrophy, follicular cell, thyroid

Other Terms

• Enlarged follicular cells

Pathogenesis

• Follicular cell hypertrophy is usually a consequence of increased TSH production by the pituitary. Exogenous (e.g., chemical) drivers of increased TSH secretion potentially involve mechanisms such as inhibition of TH synthesis, increased TH metabolism, interference with T3 to T4 conversion, interference with TH transport protein (i.e., transthyretin) binding, chemical binding to TH receptors, and autoimmunity³⁸.

Qualifiers (secondary descriptors)

• Ectopic

Diagnostic Features

• Cytoplasmic enlargement of follicular epithelial cells from flattened or cuboidal to columnar

• Occasionally see enlarged cuboidal cells rather than columnar

• Nucleus typically in basilar location

Differential Diagnoses

• Follicular cell hyperplasia

• Follicular cell adenoma

Comments

Unfortunately, the fish literature contains examples in which the tangentially-sectioned thyroid follicles have been misdiagnosed as hypertrophic (for example see Liu et al.³⁹). Note that the thyroid follicular epithelial cells of male medaka typically appear hypertrophic relative to those of cohort females⁴⁰, which seems to be a secondary sex characteristic; consequently, changes in thyroid follicular cell conformation may possibly be influenced by reproductive hormones (or mimics) in that species.

Nomenclature recommendation

Hypertrophy often accompanies follicular cell hyperplasia, although this is not always the case, and therefore these diagnoses may be made independently where the pathologist deems this to be appropriate. For convenience, and depending on the particular study results, follicular cell hypertrophy may be combined with follicular cell hyperplasia as the combined term: “follicular cell hyperplasia / hypertrophy.”

Thyroid - Proliferative Non-Neoplastic

Hyperplasia, follicular cell, thyroid

(Figs. 4.45, 4.46, 4.47 and 4.48)

Fig. 4.45.

Zebrafish, female. Thyroid. Hyperplasia, follicular cell. Extensive hyperplasia can create mass-like lesions that protrude ventrally from the oropharynx, which is a common presentation for many species. H&E.

Fig. 4.46.

Zebrafish. Thyroid. Hyperplasia, follicular cell (higher magnification of Fig. 4.45). H&E.

Other Terms

• Increased follicular cells

Pathogenesis

• Follicular cell hyperplasia is usually a consequence of increased TSH production by the pituitary. Exogenous (e.g., chemical) drivers of increased TSH secretion potentially involve mechanisms such as inhibition of TH synthesis, increased TH metabolism, interference with T3 to T4 conversion, interference with TH transport protein (i.e., transthyretin) binding, chemical binding to TH receptors, and autoimmunity³⁸

Modifiers (qualifiers or secondary descriptors)

• Ectopic

• Nodular

• Diffuse

Diagnostic Features

• Increased numbers of follicles and/or follicles lined by increased numbers of follicular epithelial cells (epithelial crowding)

• Follicles remain well-differentiated, and lined by a single epithelial cell layer

• Follicular epithelial cells may remain flattened in some cases or they may be hypertrophic (i.e., cuboidal to columnar, and they are often basophilic)

• Minimal papillary infolding may be present

• The amount of colloid may be abundant (e.g., “colloid goiter”), variable, or markedly reduced

• Growth patterns can be nodular (lobular), but usually do not form a discrete expansile mass

• Mitotic figures are rare

• Appearance of localized invasion (e.g., of bone, muscle, etc.) is attributable to hyperplastic growth of ectopic rests rather than metastasis

Differential Diagnoses

• Follicular cell adenoma – well circumscribed space occupying mass with or without compression

• Follicular cell carcinoma – prominent pleomorphism with an anaplastic growth pattern and decreased or absent colloid

Comments

Ectopic follicular cell hyperplasia was mistaken for malignant metastasis in the early literature. Note that the thyroid follicular epithelial cells of male medaka typically appear hyperplastic relative to those of cohort females.

Nomenclature recommendation

Hyperplasia often accompanies follicular cell hypertrophy, although this is not always the case, and therefore these diagnoses may be made independently. In some circumstances and depending on the particular study results, follicular cell hyperplasia may be combined with follicular cell hypertrophy as the combined term: “follicular cell hyperplasia / hypertrophy.”

Thyroid - Proliferative Neoplastic

Adenoma (B), follicular cell, thyroid

(Figs. 4.49, 4.50, and 4.51)

Fig. 4.49.

Zebrafish. Thyroid. Adenoma, follicular cell, and hyperplasia, follicular cell (around the adenoma). Located in the ventral jaw region of an adult male zebrafish. H&E.

Fig. 4.50.

Zebrafish. Thyroid. Adenoma, follicular cell (higher magnification of Fig. 4.49). The adenoma is in the upper left whereas the hyperplastic tissue is in the lower right. H&E

Fig. 4.51.

Medaka. Thyroid. Adenoma, follicular cell, and hyperplasia, follicular cell. Mass is located dorsal to the ventral aorta. Some adenomas such as this may have large cystic spaces (cystadenoma). There is a narrow ribbon of hyperplastic thyroid tissue between the adenoma and the aorta. H&E. Image courtesy of Jeffrey Wolf.

Other Terms

• Benign follicular cell tumor

Pathogenesis

Because they often appear within areas of follicular cell hyperplasia, it has been speculated that follicular cell neoplasms may have a tendency to arise from hyperplastic lesions³⁵; presumably this is due to the increased opportunity for transcription errors in a cell population characterized by increased cell division. Carcinogenic chemical induction of thyroid follicular cell tumors may occur in addition to, or in lieu of, hyperplasia^{41, 42}

Modifiers (qualifiers or secondary descriptors)

• Solid

• Papillary

• Cystic

Diagnostic Features

• Well-circumscribed, discrete, expansile mass

• Often, but not always, found within areas of follicular cell hyperplasia

• Follicles are moderately well-differentiated (still recognizable as thyroid gland) and lined by a single layer of follicular epithelial cells

• Cells are usually hypertrophic, and papillary infolding may be prominent

• Adenomas are internally homogeneous, but the growth pattern and/or coloration typically differs from the surrounding hyperplastic thyroid tissue (if present)

• The amount of colloid is usually variable to markedly reduced, in which case the lesion may have a nearly solid appearance

• Mitotic figures are rare

Differential Diagnoses

• Follicular cell hyperplasia may be focal or multifocal, can mimic invasiveness

• Follicular cell carcinoma – prominent pleomorphism with an anaplastic growth pattern and decreased or absent colloid

Comments

Adenomas differ from hyperplasia because the former are discrete expansile masses that tend to be less well-differentiated compared to hyperplastic lesions. Carcinomas do not retain the basic follicular architecture in which follicles are lined by a single cell layer, are generally not discrete lesions, and exhibit cellular atypia and increased numbers of mitoses³⁵.

Carcinoma, (M) follicular cell, thyroid

(Figs. 4.52, 4.53, 4.54, and 4.55)

Fig. 4.52.

Zebrafish, male. Thyroid. Carcinoma, follicular cell. H&E.

Fig. 4.53.

Zebrafish, male. Thyroid. Carcinoma, follicular cell (higher magnification of Fig. 4.52). The thyroid tissue origin of this poorly-differentiated tumor is barely perceptible. H&E.

Fig. 4.54.

Zebrafish, female. Thyroid. Carcinoma, follicular cell. This tumor displays greater cellular atypia than the carcinoma in the preceding figure, however a couple of rudimentary follicles are evident. H&E.

Fig. 4.55.

Zebrafish, female. Thyroid. Carcinoma, follicular cell. A different area of the carcinoma in Fig. 4.54. Mitotic figures are apparent at a frequency of approximately 2–3 per 40× objective field. H&E.

Other Terms

• Malignant follicular cell tumor

Pathogenesis

Because they often appear within areas of follicular cell hyperplasia, it has been speculated that follicular cell neoplasms may tend to arise from hyperplastic lesions³⁵; this is presumably due to the increased opportunity for transcription errors in a cell population characterized by increased cell division. Carcinogenic chemical induction of thyroid follicular cell tumors may occur in addition to, or in lieu of, hyperplasia^{41, 42}

Modifiers (qualifiers or secondary descriptors)

• Well differentiated

• Poorly differentiated

Diagnostic Features

• Small to very large, poorly circumscribed, ovoid or irregular mass

• Often, but not always, found within areas of follicular cell hyperplasia

• Generally, see piling up of cells rather than follicles lined by a single cell layer

• Even relatively “well-differentiated” tumors do not usually display standard thyroid follicular architecture

• In poorly differentiated carcinomas, evidence of follicle formation may be minimal or not at all evident

• Frequently, minimal if any colloid formation

• Common to see marked cellular atypia and numerous mitotic figures

Differential Diagnoses

• Follicular cell hyperplasia may be focal or multifocal, can mimic invasiveness

• Follicular cell adenoma is a well circumscribed space occupying mass with or without compression

Comments

Although local invasion and distant metastasis are the hallmarks of malignant thyroid tumor identification in mammals, these attributes are not generally useful for fish thyroid neoplasms because of the widespread distribution of ectopic thyroid progenitor tissue in fish. Instead, diagnoses of malignancy are based primarily on aberrant growth patterns and cytologic atypia. Additionally, there is evidence in both fish and rodents that even true thyroid neoplasms may regress following correction of the stimulus for cell proliferation (e.g., low iodine)^{43, 44}. One proposed theory to explain this is that tumors generated during prolonged periods of elevated TSH production may remain TSH-dependent due to the lack of selection pressure to become TSH-independent⁴⁴.

7. Ultimobranchial gland

The ultimobranchial gland (body) is a single midline organ located dorsal to the heart within the transverse membrane that separates the pericardial cavity from the coelomic cavity. This finely- encapsulated angular organ consists of deeply basophilic cells that have basally oriented nuclei and are arranged in pseudorosette patterns. Small and often difficult to capture in histologic sections, the ultimobranchial body is readily visualized in zebrafish which, coincidentally, is also the species in which neoplasms of this gland are most likely to be found⁴⁵. Fish do not possess thyroid gland C-cells, and instead calcitonin is secreted primarily by the ultimobranchial gland.

There are several reports of toxicologically-induced lesions involving the ultimobranchial gland, however most are not highly credible based on the suboptimal quality of the histologic sections (as evident in the photomicrographic Figs. 4.56 and 4.57) and the associated diagnostic interpretations in several publications^46,47,48,49.

Fig. 4.56.

Zebrafish, male. Ultimobranchial gland. Normal (rectangle). Gland is located ventral to the esophagus and dorsal to the atrium. H&E. Image courtesy of Jeffrey Wolf.

Fig. 4.57.

Zebrafish, male. Ultimobranchial gland. Normal (arrow) (higher magnification of Fig. 4.56). The secretory cells are arranged as pseudorosettes. H&E. Image courtesy of Jeffrey Wolf.

Table 7 indicates nomenclature, diagnostic criteria, and differential diagnoses partaining to the ultimobranchial gland.

Table 7. Nomenclature, diagnostic criteria, and differential diagnosis of ultimobranchial gland.

Ultimobranchial Gland		Common	Uncommon	Not observed, but potentially relevant	Not applicable
Non-proliferative lesions
	*Degeneration		x
Proliferative
	Neoplastic
	*Adenoma		x
	*Carcinoma		x

*Terminology with diagnostic criteria or comments described in the main text.

Ultimobranchial Gland - Congenital Lesions

Congenital lesions of the ultimobranchial gland are not typically observed in toxicological studies although cysts may be observed.

Ultimobranchial Gland - Non-Proliferative

Degeneration, ultimobranchial gland

(Fig. 4.58)

Fig. 4.58.

Zebrafish. Ultimobranchial gland. Degeneration, cystic. H&E

Other Terms

• None

Modifiers

• Cystic

Pathogenesis

• This lesion has been observed as an age-related change in zebrafish

Diagnostic Features

• Single chamber or multilocular

• Flattened epithelial or endothelial lining

Differential Diagnoses

• Congenital (ultimobranchial gland/body) cyst

• Necrosis

Ultimobranchial Gland - Proliferative Non-Neoplastic

Most proliferative ultimobranchial lesions are considered to be neoplastic (see Ultimobranchial gland, Adenoma and Carcinoma).

Ultimobranchial Gland - Proliferative Neoplastic

Adenoma, (B) ultimobranchial gland

(Fig. 4.59 and 4.60)

Fig. 4.59.

Zebrafish. Ultimobranchial gland. Adenoma. H&E.

Fig. 4.60.

Zebrafish. Ultimobranchial gland. Adenoma (higher magnification of Fig. 4.59). H&E.

Other Terms

• Benign ultimobranchial tumor

Pathogenesis

• It has been speculated that husbandry factors (e.g., elevated dietary calcium) may play a role⁵⁰

• Rare ultimobranchial gland adenomas were diagnosed in zebrafish twelve months after their exposure to N-methyl-N’-nitro-N-nitrosoguanidine (MNNG)⁵¹

• Diagnostic Features

• Well-circumscribed expansile mass

• Epithelial cells form ribbons and partial pseudorosettes

Differential Diagnoses

• Ultimobranchial carcinoma

Comments

Ultimobranchial gland hyperplasia has not been described, and therefore, proliferative ultimobranchial gland lesions are generally diagnosed as adenomas or carcinomas. These tumors are relatively common in captive zebrafish greater than 1.5 years of age, although they can be seen as early as 6 months in particularly susceptible lines (e.g., TL strain)⁵². Adenomas differ from carcinomas by virtue of local invasiveness demonstrated by the latter. They have also been observed in aged male broodstock that had seminomas⁵³.

Carcinoma, (M) ultimobranchial gland

(Figs. 4.61, Figs. 4.62, 4.63)

Fig. 4.61.

Zebrafish. Ultimobranchial gland. Carcinoma. H&E.

Fig. 4.62.

Zebrafish. Ultimobranchial gland. Carcinoma (higher magnification of Fig. 4.61). Malignancy is indicated by penetration of the neoplastic tissue through the gland capsule (left side) and tumor extension into the lumen of the neighboring sinus venosus. H&E.

Fig. 4.63.

Zebrafish. Urophysis. Normal. Located at the terminus of the spinal cord near the tail. H&E. Image courtesy of Jeffrey Wolf.

Other Terms

• Malignant ultimobranchial tumor

Pathogenesis

• It has been speculated that husbandry factors (e.g., elevated dietary calcium) may play a role⁵⁰

Diagnostic Features

• Nests, ribbons, and pseudorosettes of amphophilic polygonal cells with basilar nuclei surrounded by a fibrovascular stroma

• Usually larger than adenomas (e.g., 50x the size of normal glands) and growth pattern is generally more disorganized

• Key to diagnosis is tumor invasion through the glandular capsule

Differential Diagnoses

• Ultimobranchial gland adenoma

Comments

Ultimobranchial gland hyperplasia has not been described, and therefore, proliferative ultimobranchial gland lesions are generally diagnosed as adenomas or carcinomas. Adenomas differ from carcinomas by virtue of local invasiveness demonstrated by the latter. These tumors are relatively common in captive zebrafish greater than 1.5 years of age, although they can be seen as early as 6 months in particularly susceptible lines (e.g., TL strain)⁵².

8. Urophysis

Characterized as a ventral swelling at the posterior end of the spinal cord, the urophysis is structurally similar to the pituitary neurohypophysis. This organ, which is unique to jawed fishes, is rarely examined in toxicologic bioassays because pathologic findings involving the urophysis have not been reported, and it is located in the tail which is not collected routinely for microscopic examination (Figs. 4.63 and 4.64).

Fig. 4.64.

Zebrafish. Urophysis. Normal (higher magnification of Fig. 4.63). This neurosecretory organ is rarely examined in fish toxicological studies. H&E. Image courtesy of Jeffrey Wolf.

Major hormones synthesized and secreted by the urophysis are urotensin I, which is structurally similar to corticotropin-releasing hormone, and urotensin II, which shares structural similarity with somatostatin⁵⁴. Urotensins are thought to influence stress responses and osmoregulation and may function as brain neurotransmitters. Urotensin II, which is also present in mammals, has vasoactive effects when administered experimentally⁵⁴.

Spontaneous or induced lesions of the piscine urophysis have not been reported.