ACKNOWLEDGEMENT AND DISCLAIMER: This is a draft chapter that has not been peer reviewed.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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Pituitary Gland Figures 1-32

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Introduction

The pituitary is functionally a master endocrine tissue involved in homeostasis and regulation of growth, reproduction, sexual maturation, pregnancy, metabolism, lactation, and stress responses. The adenohypophysis includes the anterior and intermediate lobes. Cellular components of anterior lobe produce growth hormone, prolactin, thyroid stimulating hormone, leutotrophic hormone, and follicle stimulating hormone that function as trophic factors for peripheral target tissues. The neurohypophysis (posterior pituitary) consisting of the pars nervosa and infundibular stalk produces oxytocin and vasopressin (antidiuretic hormone). The two parts of the pituitary are functionally controlled by the hypothalamus (hypothalamic-pituitary axis) and feedback mechanisms from hormonal target tissues. Studies during the past decade using genetically modified mice have provided significant understanding of human pituitary dysfunction and provided valuable insight into the importance of pituitary progenitor/stem cells and the plasticity of pituitary responses in maintaining homeostasis.

Embryology

The mouse pituitary gland (hypophysis) consists of an anterior and posterior lobe. As with other endocrine organs, there is separate embryological origin of the two components. The anterior pituitary (adenohypophysis) originates from Rathke’s pouch (an oropharyngeal diverticulum) while the posterior pituitary (neurohypophysis) forms from neuroectoderm on the floor of the 3rd ventricle (diencephalon) (Kerr 1946; Kaufman 1994; Kaufman et al., 2010). In the mouse, the placode that forms Rathke’s pouch (RP) is present at 7.5 days post coitum (dpc) with formation of a rudimentary RP at approximately 8.5 to 9 dpc (Kelberman et al., 2009; Dubois & El Amraoui 1995). A day later an infundibulum recess develops from neuroectoderm on the floor of the 3rd ventricle (ventral diencephalon) to ultimately become the posterior pituitary and pituitary stalk. This neuroectoderm forms in juxtaposition with RP.

The juxtaposition of the neural and oral ectoderm is maintained throughout subsequent development of the pituitary and allows for inductive interaction during development and formation of the anterior and posterior pituitary (Takuma et al., 1998; Kelberman et al., 2007). Multiple transcription factors and genes are involved in the highly coordinated sequential steps in cellular commitment and positioning of pituitary cell lineages, all responsive to intrinsic and extrinsic signaling gradients (Cushman et al. 2001; Dasen & Rodenfeld 2001; Dasen & Rosenfeld 1999; Asa & Ezzat 1999; Kelberman et al. 2009; Rizzoti 2015). Corticotrope, thyrotrope, somatotrope, lactotrope and finally gonadotrope cells develop in association with specific gene expression between 12.5 and 16.5 dpc (Kelberman et al., 2009). Starting at about 14 dpc, the internal carotid artery provides branches to the hypothalamic area to ultimately become the hypophyseal portal system, the main blood supply to the anterior pituitary (Kaufman et al., 2010). The hypophysis and pituitary stalk receive blood from superior hypophyseal arteries. Starting at about 15 dpc, progressive rapid proliferation of anterior and posterior pituitary and the vascular supply commences, along with obliteration of the lumens of PR and posterior pituitary and early ossification of the sella turcica. Published histologic features showing progressive pituitary gland development up to approximately 18 dpc are clearly shown in stained sections (Kaufman et al., 2010). At this stage of development there is merging of anterior and posterior pituitary with compression of RP and further maturation of the pituitary gland. Ultrastructural demonstration of granular cells producing thyroid stimulating hormone (TSH), follicle stimulating hormone (FSH), leutinizing hormone (LH), and somatotrophic hormone (STH) is present at gestation day 16 in SMA mice following by a proportional increase in STH producing cells (Sano & Sasaki 1969). Throughout the process of pituitary organogenesis there is active migration of progenitor cells, cell patterning and cell differentiation under the influence of multiple growth and transcription factors (Zhu et al., 2007; Dasen et al., 1999; Dasen et al., 2001; Rizzoti & Lovell-Badge 2005). After birth, there is continued proliferation and differentiation of hormone-producing cells as well as establishment of stem/progenitor cells (Levy 2002; Levy 2008; Nolan et al., 1998).

Anatomy

The mouse pituitary is located in a midline shallow depression, the sella turcica, on the dorsal surface of the basiphenoid skull bone. A dual tissue origin results in an anterior (adenohypophysis) and posterior (neurohypophysis) pituitary gland that fuse during embryogenesis with the point of fusion becoming the pars intermedia. The adenohypophysis represents the major portion of the pituitary gland and consists of the pars distalis (pars anterior), the pars intermedia, and the pars tuberalis. A cleft remnant of Rathke’s pouch is located between the pars distalis and pars intermedia. The neurohypophysis (pars posterior) includes the median eminence of the tuber cinereum, infundibular stalk, and pars nervosa. The neurohypophysis connects to the hypothalamus by the infundibular stalk. The small pars tuberalis partially wraps around the infundibular stalk.

When the brain is removed at necropsy, the pituitary stalk attachment to the brain is severed. Since the mouse pituitary is small and fragile, it is often fixed in situ for subsequent histologic examination. The histological preparation is typically a coronal section showing the pars distalis, pars intermedia and pars nervosa without the infundibular stalk and pars tuberalis. The pituitary is larger and heavier in females with weight differences among mouse strains (Liebelt 1994).

Histology

Adenohypophysis

The bilobed pars distalis, the largest component of the adenohypophysis, surrounds the pars intermedia and pars nervosa and is comprised of nests and cords of round to polygonal cells interspersed within a rich fibrovascular plexus. In hematoxylin and eosin-stained sections, pars distalis cells are chromophilic being tinctorially acidophilic or basophilic or alternatively are chromophobic (Figures 1 & 2). Chromophilic cells contain secretory cytoplasmic granules that account for their tintorial staining. Acidophilic chromophils are characterized by small round or oval cells with a central nucleus while basophilic chromophils are more polyhedral with an eccentric nucleus (Leibert 1994). Chromophobic cells have a large nucleus with 1 or 2 nucleoli, lack obvious secretory granules, and may serve as resting reserve cells or simply be in an early active synthesis phase. Immunohistochemistry has been used to more definitively identify specific cell types in the adenohypophysis (Sasaki & Iwama 1988; Sano & Sasaki 1969; Capen et al., 1991; Liebelt 1994). Several distinctive cell types of the mouse adenohypophysis have been identified by electron microscopy (Liebelt 1994). Histomorphology as well as ultrastructural cytologic features may be influenced by sex and the physiological state of the pituitary. Sex differences in the proportions of lactotrophs and somatotrophs have been documented by immunohistochemistry and electron microscopy in SMA mice along with documentation of presence of a few mammosomatotrophs (Sasaki & Iwama 1988).

The proportion of each cell type varies dynamically depending upon physiological conditions. Pars distalis cells are responsible for secretion of major pituitary hormones and include somatotrophs that secrete growth hormone and mammotrophs that secrete prolactin in females. Somatotrophs and mammotrophs are typically acidophilic in routine H&E stains. Basophilic gonadotrophs produce follicle stimulating hormone and leutinizing hormone, are more numerous in males than in females, but their frequency in females varies depending upon the estrus cycle stage. Thyrotrophs are also basophilic and secret thyroid stimulating hormone. However, tinctorial features of pituitary cells, especially in the pars distalis, may not accurately reflect phenotype and cell type frequency. Using immunohistochemistry, Baker and Gross concluded that the frequency and distribution of hormone producing cells in the pars distalis of Swiss-Webster mice were similar to that in other species (Baker & Gross 1978).

The adult rodent pituitary has a low steady-state cell turnover under both hypothalamic neuroendocrine control and feedback control from peripheral target tissues (Fauquier et al. 2008; Nolan et al. 1998). Dynamically changing pituitary hormonal responses to changing physiology are controlled by a complex constellation of molecular transcription factors and genes during development and in adulthood as well as during pituitary neoplasia (Zhu et al. 2007; Asa & Ezzat 1999; Sav 2014). The original notion that different chromophilic pituitary cells were each specialized to only produce a specific hormone is no longer valid. Pituitary cells are plurihormonal and proportions of trophic cells in mice vary under differing conditions as a reflection of pituitary plasticity. Examples include increases in lactotrophs in nursing mice, strain difference in high and low incidence mammary tumor strains (Sasaki et al. 1978); increased gonadotrophs following castration in mice (Levy 2008; Inoue & Kurosumi 1981); and increase in corticotrophs in cold stressed male and female mice (Senovilla et al. 2008). We now better appreciate the importance of pituitary flexibility in adjusting its hormone producing repertoire in response to changing physiological dynamics, including pregnancy, lactation, metabolism, stress, and age.

The plasticity of pituitary hormonal response comes from recruitment of uncommitted chromophobe cells (Levy 2002), transdifferentiation of committed cells (Nunez et al. 2003; Vidal et al. 2001), and/or cell division (Levy 2002) but for rapid responses to changing physiological conditions, cell proliferation is not likely to be a major contributor (Nolan et al 1998; Levy 2008). Also contributing to pituitary plasticity in response is recruitment of cells from the stem/progenitor cell compartment (Vankelecom 2010; Rizzoti et al. 2013). It is estimated that 1.5% of mouse anterior pituitary cells are multipotent progenitor/stem cells (Florio 2011). SOX2-expressing progenitor cells responding to hormonal demands are capable of generating all major cells types in the adult mouse pituitary. SOX2+/SOX9+ identifies transit-amplifying cells and SOX2+/SOX9- cells are multipotent progenitor/stem cells (Fauquier et al. 2008; Florio 2011).

Among the chromophobic cells of the pars distalis is a small population of non-secretory folliculo-stellate cells that have properties of dendritic cells and are variably immunoreactivity for S-100 protein. Their phenotype suggests a lymphoid/mesenchymal anlage in pituitary development in addition to the known neuroectoderm and oral ectoderm tissue anlage (Allaerts et al. 1991; Allaerts et al. 1993; Vankelecom et al. 1993).

Cells of the pars intermedia are larger than those of the pars distalis, are polygonal with densely stained nuclei, and are arranged in groups separated by connective tissue and vascular channels (Liebelt 1994)(Figures 1 & 2). Using fluorescent immunohistochemistry, a-melanocyte stimulating hormone (MSH), b-MSH and adrenocorticotrophic hormone (ACTH) producing cells have been identified in the pars intermedia using fluorescent immunohistochemistry with enhanced excretion by these cells after dehydration and adrenalectomy (Roux & Dubois 1976). The pars intermedia and pars nervous are separated by a capillary plexus with contact zones where the pars intermedia and pars nervosa have direct communication (Jarskar 1977).

There is increasing evidence that a subpopulation of non-hormonal cells function as stem/progenitor cells in the embryonic and adult mouse pituitary gland. These stem cells express high-mobility group (HMG) SOX2 and SOX9 transcription factors and are located around the pituitary cleft (remnant of Rathke’s pouch) as well as scattered in the anterior lobe (Rizzoti 2015; Rizzoti et al., 2013; Andoniadou et al., 2013; Vekelecom 2010; Fauquier et al., 2008). They actively migrate during embryonic and postnatal pituitary organogenesis and in the adult mouse reside in niches where they maintain their stemness (Yoshida et al., 2016).

Neurohypophysis

The neurohypophysis consists of the pars nervosa, the infundibular stalk, and the median eminence of the tuber cinerum of the hypothalamus. The neurohypophysis is hypocellular and mostly occupied by unmyelinated axons of neurosecretory magnocellular neurons originating in the supraoptic and paraventricular nuclei of the hypothalamus (Kelberman et al., 2009) (Figures 1 & 2). The axons terminate in association with a rich capillary network that is supported by modified glial cells (pituicytes). Precursors for the hormones oxytocin and vasopressin are synthesized in the hypothalamic supraoptic and paraventricular nuclei, transported along the axons, stored in axon terminals for subsequent cleavage into active hormone with ultimate release into the vascular capillary network.

Vascular supply

Capillary networks develop in the mouse embryo pituitary as early as gestation day 16 and continue to extend into the parenchyma throughout embryogenesis (Hashimoto et al., 1998). Blood from branches of the internal carotid arteries and the circle of Willis form narrow capillaries in the posterior lobe with the pars intermedia being relatively avascular. A rich blood supply penetrates the pars tuberalis that provides a scaffold for a capillary plexus starting at the median eminence with ultimate drainage into the hypophyseal portal plexus that supplies the pars distalis (Kaufman et al., 2010; Capen 1996).

Physiology

Sometimes referred to as the master gland, the pituitary is controlled by the neurosecretory neurons in the hypothalamus that produce small peptides called releasing hormones and releasing-inhibitory hormones that enter the hypophyseal portal system to act on specific cell types in the adenohypophysis. The releasing hormones bind to specific receptors to initiate Ca2+ signals and subsequent secretion of the appropriate trophic hormone that is specific for a given target tissue. The whole endocrine system is kept in balance by a feedback mechanism whereby the blood concentration of a specific target tissue hormone acts on the hypothalamus to up- or down-regulate production of releasing or releasing-inhibitory signals from the hypothalamus. As examples, TSH is inhibited by T3/T4 from the thyroid; FSH and LH by androgens and estrogens; ACTH by adrenal corticosteroids; and growth hormone (GH), prolactin and MSH by their corresponding release inhibiting hormones.

Anterior Pituitary

Pars Distalis

The anterior pituitary secretes seven endocrine hormones controlled by releasing and inhibitory factors (Table 1): prolactin from mammotrophs, luteinizing hormone and follicle-stimulating hormone from gonadotrophs, growth hormone from somatotrophs, thyroid stimulating hormone from thyrotrophs, adrenocorticotrophic hormone from corticotrophs, and melanocyte-stimulating hormone from melanotrophs. The proportion of the various hormone producing trophic cells fluctuates as a dynamic reflection of changing physiology (Levy 2002). For example, during lactation numerous acidophils and chromophobe cells may be produced with the acidophils generated primarily from transition of chromophobe cells and to a much smaller extent from cell division. Other types of cells show differences associated with sexual maturity (Yamada et al., 1957). Strain differences in number and size of prolactin secreting cells along with differences in ultrastructural features suggest that secretory activity in prolactin secreting cells is higher in diestrus female C3H than in C57BL mice (Sasaki et al. 1979). Cortisol injections in sexually mature C57BL/6J mice results in reduced numbers of mammotrophs and increased numbers of somatotrophs while adrenalectomy is associated with increases in mammotrophs and decreases in somatotroph numbers (Keough and Wood 1978). Pulsatile hormone release to control homeostasis in response to changing physiology and metabolism also likely involves modulation of blood flow (Schaeffer et al. 2010).

Table 1. Pars Distalis Cells and Their Controls.

Folliculo-stellate cells located in the adenohypophysis form a functional network of intrapituitary communication. The narrow cytoplasmic processes of folliculo-stellate cells extend between glandular cells providing circuitry than can transmit action potential rises in cytoplasmic Ca2+ signals to relay information across different regions of the pituitary gland and thereby fine tune highly orchestrated pituitary function (Fauquier et al. 2001). Folliculo-stellate cells express S-100, share similarities with CNS glial cells, and are immunopositive for dendritic cell aminopeptidase (Allaerts et al. 1991). They produce bioactive interlukin-6 to regulate cell division and hormone synthesis, thereby further establishing cross-talk between the immune and neuroendocrine systems (Vankelecom et al. 1993). While leptin is expressed primarily in thyrotrophs, it is also expressed in folliculo-stellate cells and these cells also have an isoform of a leptin receptor suggesting a possible autocrine/paracrine loop in the pituitary (Jin et al. 2000).

Pars Intermedia

The pars intermedia contains polygonal cells with pale staining foamy cytoplasm and is separated from the pars distalis by a cleft, a remnant of Rathke’s pouch (Figures 1 & 2). The mouse pars intermedia is larger than in other species and is the source of adrenocorticotrophic hormone, melanocyte stimulating hormone and beta-endorphin (Oishi et al., 1992). ACTH-secreting corticotrophs represent the major cell type in the pars intermedia, although a few corticotrophs may also be present in the pars distalis. Following adrenalectomy in the mouse, the pars intermedia becomes hyperactive with greater excretion of MSH and ACTH-like polypeptides (Roux and Dubois 1976). The pars intermedia is in direct contact with the pars nervosa being separated only by a capillary plexus without a basement membrane (Eurenius and Jarskar 1975) allowing intimate contact between these two parts of the pituitary. Adrenergic nerve terminals in the pars intermedia have synaptic-like contact with pars distalis glandular cells suggesting there is some direct nervous control in addition to humoral trophic effects on the pars intermedia (Jarskar 1977). MSH, ACTH and a corticotropin-like peptide have been demonstrated in the pars intermedia (Jarskar 1977). Despite these features of the pars intermedia, its exact functional significance requires further clarification.

Pars Tuberalis

The pars tuberalis is a main target of melatonin with a high density of the MT1 major melatonin receptor and thereby acts as an important interface in transmitting photoperiod information to the endocrine system (von Gall et al. 2005; Unfried et al. 2009). The melatonin action on the MT1 receptor leads to rhythmic expression of the clock genes (mPERa, mPER2, mCRY1), thereby driving prolactin secretion from the pars distalis (Morgan and Williams 1996; Jilg et al. 2005).

Posterior Pituitary

The unmyelinated axons of magnocellular secretory neurons located in the paraventricular and supraoptic nuclei of the hypothalamus transport secretory material to their terminal ends. The secretory material accumulates as variably sized spherical bodies known as Herring bodies and consists of molecular precursor molecules and neurophysin carrier protein that when cleaved will release active oxytocin and vasopressin into the associated vascular capillary network. Release of oxytocin and vasopressin is modulated by pituicytes that surround the neurosecretory axons and axon terminals with their cellular processes extending between the secretory endings and the basal lamina of fenestrated capillaries. Hormone release is under osmotic and receptor stimulated control and results in retraction of pituicyte processes, permitting increased contact of axon endings and capillaries (Hatton 1988). Triggering of oxytocin and vasopressin release by magnocellular secretory neurons relies on integration of reproductive, cardiovascular and osmotic afferent signaling as well as on regulatory features intrinsic to the magnocellular neurons themselves (Brown 2016). Vasopressin maintains bodily fluid balance and blood pressure and has vasoconstrictive properties. Oxytocin is important for parturition and lactation but has a large repertoire of many other activities and also acts in a neuroendocrine fashion to control or modulate release of prolactin and ACTH and possibly also gonadotrophins (Samson and Schell 1995).

Non-proliferative Changes

Pituitary Cysts

Pituitary cysts are incidentally seen in chronic mouse studies and may occur in the pars distalis and intermedia and rarely in the pars nervosa (Blumentahl 1955; Mahler & Elwell 1999; Liebelt 1994; Morton & Tekeli 1997). A 10 to 20% incidence has been documented in one study (Watanabe 1991). They may be associated with Rathke’s cleft but can occur randomly. The epithelial lining of unilocular or multilocular cysts may be cuboidal or columnar with or without cilia or irregularly lined by resident parenchymal cells. Their epithelial lining distinguishes them from angiectasis or dilated vascular channels. They are distinguished from cytic degeneration in that in the latter case spaces are lined by degenerating secretory cells. In rare cases cysts may have a squamous epithelial lining, suggesting thyroglossal duct origin. Cysts are typically filled with homogeneous or finely granular eosinophilic proteinaceous material (Figures 3 & 4). While cysts are presumptive developmental changes representative of the craniopharyngeal Rathke’s pouch origin, the possibility of cysts developing in mature mice cannot be excluded. Except in very unusual situations, it is not usually necessary to subclassify cysts based on presumed site of origin.

Degenerative Changes and Cytological Alteration

Truly degenerative pituitary lesions are not common in mice. Ablation of pituitary hormone target tissues including adrenal, thyroid, and gonads results in a hypertrophic and proliferative response in pituitary corticotrophs, thyroidtrophs, and gonadotrophs, respectively. In the case of gonadectomy in Wistar rats, the resulting gonadotrophs become enlarged and vacuolated and are referred to as “castration cells” (Inoue and Kurosumi 1981) and a similar response would be expected in mice. The increased volume of castration cells in rats is associated with increased rough endoplasmic reticulum, dilated endoplasmic reticulum cisternae, and increased numbers of secretory granules. Castration cells in mice are typically less prominent and less vacuolated compared to these cells in rats (Ranadive and Karande 1970). Development of “exhaustion cells” and “castration cells” are responses to changes in systemic physiology and could be considered adaptive changes rather than degenerative changes (Mahler and Elwell 1999). Mouse strain differences in gonadotroph responses following ovariectomy were earlier and more pronounced in C3H than in ICRC and C57 mice (Ranadive and Karande 1970).

Cystic degeneration, also referred to as cystoid degeneration, of the pars distalis may be seen in aging control mice as well as following treatment of female C3H HeN (MMTV+) with natural or synthetic estrogens (Cameron & Sheldon 1996; Firth & Ward 1988; Liebelt 1994). Cystic degenerative lesions lack an epithelial lining but rather are lined by parenchymal pituitary cells, some of which may show degenerative changes. Cytic degenerative lesions contain eosinophilic material with or without cellular debris (Figures 5 & 6).

Inflammatory and Vascular Changes

Inflammation restricted to the mouse pituitary is uncommon but may be seen as part of a more generalized process such as part of meningitis. Treatment induced hypophysitis and associated hemorrhage and atrophy is reported in SLJ/J female mice immunized with complete Freund’s adjuvant (Lupi et al. 2011). Following immunization with mouse pituitary extract, severe lymphocytic autoimmune hypophysitis with multinucleated cells was produced in SJL (Tzou et al. 2008). The pituitary gland is highly vascular and small areas of angiectasis may be seen as a common background change and are usually not diagnosed if of minimal severity.

Hypertrophic Lesions

Hypertrophy of the pars distalis and pars intermedia is usually a negative feedback response secondary to decreased function of target endocrine tissues. Hypertrophy may occur in combination with hyperplasia. Pars distalis hypertrophy is usually bilateral, may be generalized or occurring as multiple small clusters, and when extensive will significantly increase the size of the pituitary. The hypertrophic cells are readily apparent by increased volume of pale staining cytoplasm (Figures 7 & 8). Focal hypertrophy of the pars distalis can occur but is less common (Figures 9 & 10). Hypertrophy of the pars intermedia (Figures 11 & 12) is uncommon but likely generalized with the enlarged cells having increased cytoplasm volume, paler than normal staining, but retaining their structural arrangement in small groups and without an increase in nuclear density.

Proliferative Lesions

In general, pituitary tumorigenesis can occur via intrinsic pituitary abnormalities such as specific somatic mutations or via an abnormal regulatory mechanism such as escape from hypothalamic inhibition (Levy & Lightman 1993). Most pituitary tumors that occur in the mouse and rat in chronic toxicity/carcinogenicity studies occur late in these studies and are preceded by hyperplasia indicating that escape from hypothalamic inhibition or excessive exposure to stimulating hormone may function as tumor promoters. Indeed, the significant decrease in pituitary adenomas in diet-restricted female B6C3F1 mice is speculated to be a consequence of decreased production of hypothalamic neurotransmitters (Sheldon et al. 1995). However, the latency for pituitary tumor development in mice suggests that multiple factors may be necessary f