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Thomas J. Steinbach, Robert. R. Maronpot, And Jerry F. Hardisty
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Rodents have been used extensively in virtually all fields of biomedical research and have been the primary species used in toxicologic and carcinogenic research. Over many years it has become obvious that some conditions and in particular some tumors in rodents have questionable relevance in humans. Some of these include peroxisome proliferatoractivator receptor-α (PPAR-α) agonist-induced liver tumors, alpha2μ-globulin-induced renal tumors in male rats, and bladder tumors induced in rats by urinary calculi. In this chapter we review the human relevance of Leydig cell tumors (LCTs), which have been induced in rodents by a number of compounds. We will consider the similarities and differences between humans and rats in the physiology of the Leydig cell (LC) and the pathology of LCTs. Most importantly, we will examine the mechanisms of action that induce LCTs in rats and humans and present data on incidence, physiology, human endocrine disease, and comparative epidemiological studies that strongly indicate LCTs in rodents, in particular the rat, are of little relevance to human health.

Development

The ontogeny of the LC can be reviewed in two basic ways: by following the stages of adult LC differentiation or by examining the two recognized generations of LCs, fetal and adult. Differentiation of the LC is typically broken down into four stages: stem LCs, progenitor LCs, immature LCs, and adult LCs. Stem LCs are spindle shaped and as a cell, which has not yet committed to a lineage of development, it does not express the LC-specific markers such as luteinizing hormone receptor or steroidogenic enzymes such as 3β-hydroxysteroid dehydrogenase. In the rat, at postnatal day 14 stem LCs begin to stain positive for 3β-hydroxysteroid dehydrogenase and are identified at this point as progenitor LCs. These cells develop from about postnatal day 14 until day 28 and begin to produce androgen. (Reviewed in Chen et al., 2009) Gene expression changes most when stem LCs develop into progenitor LCs, whereas differences in gene expression from progenitor to immature LCs and from immature to adult LCs are minimal, suggesting these cells are relatively more similar (Stanley et al., 2011).

Starting at postnatal day 28, progenitor LCs transform morphologically to become more round. The immature LC has increased amounts of smooth endoplasmic reticulum and steroidogenic enzyme levels. Testosterone is not yet the major product produced by these cells because they possess high levels of androgen-metabolizing enzymes that produce 5α-androstane-3α, 17β-diol. The immature LC population doubles once from postnatal day 28 to 56, at which point they develop into adult LCs. The androgen-metabolizing enzyme activity reduces while synthesis of testosterone increases. By day 90, testosterone production in an adult LC of a rat is 150 times than that of a progenitor, and five times than that of an immature LC (Shan et al., 1993).

Development of LCs can also be discussed in reference to the two generally recognized generations: fetal and adult. In the rat, the fetal LC begins to produce testosterone at about gestation day 16 and peaks on day 19 (Habert and Picon, 1984). In humans, testosterone peaks at the end of the 1st trimester (Reyes et al., 1974). The production of androgens is critical for masculinization of the fetus during what is termed the “masculinization programming window.” In the rat this window is from 15.5 to 17.5 days of gestation while in the human it extends from 8 to 14 weeks of gestation. (Welsh et al., 2008) In addition to androgens, fetal LCs produce insulin-like growth factor 3, which is responsible for testicular descent (Zimmerman et al., 1999).

A number of factors that influence the differentiation of the fetal LCs have recently been identified and include: desert hedgehog, a cell signaling molecule secreted by Sertoli cells, and transcription factors such as GATA-4. Interestingly, in rodents, development of fetal LCs is not dependent on pituitary luteinizing hormone (LH) (until the hypothalamic–pituitary– gonadal axis begins functioning near birth). Primates, including humans, have a brief period of independence from hormonal stimulation but then fetal LCs become dependent on placental chorionic gonadotropins (Reviewed in O’Shaughnessy and Fowler, 2011; Svechnikov et al., 2010).

The adult LC forms mostly during puberty and produces the testosterone responsible for spermatogenesis, along with differentiation of other secondary sex characteristics. In both the rat and human there is decreased testosterone production associated with aging. In humans the decreased testosterone is associated with increased levels of LH and appears to be due to a decline in the number of LCs through degeneration. In rats, however, decreased testosterone is associated with declining LH levels and seems to be related to the inability of the LC to respond to LH stimulation (Reviewed in Chen et al., 2009; Cook et al., 1999).

Steroidogenesis

The LC is responsible for producing virtually all of the steroids of the testis; testosterone being the major steroid (Stocco, 1996). Although a number of pathways have been elucidated recently, the primary signaling method for steroidogenesis is initiated when LH binds to a G proteincoupled receptor, which in turn activates adenylate cyclase, producing cAMP (See Figure 109.1). Protein kinase A is activated by cAMP and results in phosphorylation of a number of proteins and transcription factors (Wang and Ascoli, 1990). One of the most important of these is steroidogenic acute regulatory protein (StAR), which is estimated to mediate 85–90% of steroid synthesis (Reviewed in Stocco et al., 2005). Steroidogenic acute regulatory protein and translocator protein, formerly known as peripheral-type benzodiazepine receptor, are components of a large, multiprotein complex known as the transducesome. This complex is responsible for transporting cholesterol from the outer mitochondrial membrane to the inner mitochondrial membrane (Reviewed in Rone et al., 2009). The translocation of cholesterol from the outer to inner mitochondrial membrane is the rate limiting step in the synthesis of all steroids (Miller, 2007; Manna 2009). Intracellular cholesterol is produced by three mechanisms: de novo synthesis in the endoplasmic reticulum; mobilization from the plasma membrane and uptake from circulating cholesterol esters; and mobilization from lipid droplets (Rone et al., 2009).

Human Relevance Of Rodent Leydig Cell Tumors-1

FIGURE 109.1 Mechanisms involved in the synthesis of testosterone.

Mediated by StAR, free cholesterol is transported to the inner mitochondrial membrane, where it is metabolized into pregnenolone by the P450 cholesterol side-chain cleavage enzyme (CYP11A1) (Reviewed in Payne and Hales, 2004). The remaining reactions of steroidogenesis occur within the endoplasmic reticulum with small differences between rats and humans in the pathway (Cook et al., 1999). In rat pregnenolone is transformed to progesterone by the enzyme 3β-hydroxysteroid dehydrogenase (3βHSD). Next the P450 enzyme, C17a-hydroxylase/C17-20-lysase (CYP17), catalyzes two reactions converting progesterone first to hydroxyprogesterone and then to C19 steroid, androstenedione. In humans, CYP17 prefers pregnenolone as a substrate and converts it to 17α-hydroxypregnenolone and then to C19 steroid, dehydroepiandrosterone (DHEA). Finally, 17β-hydroxysteroid dehydrogenase (17βHSD) converts the C19 steroid into testosterone (Payne and Hales, 2004). Testosterone can be further metabolized into estrogen by P450 aromatase (CYP19) (Payne and Hales, 2004) or into the more potent androgen dihydrotestosterone (DHT) (Cook et al., 1999).

As mentioned, cAMP-dependent activation of protein kinase A is essential for activation of StAR and other proteins and transcription factors involved in steroidogenesis, to include cholesterol esterase (CEH), steroidogenic factor-1 (sf-1), GATA-4, and cAMP response element-binding protein (CREB). However, there are other mechanisms by which steroidogenesis is regulated. In recent years research has focused on factors that influence and regulate StAR, since it is critical to the rate-limiting step of steroidogenesis. A number of factors can regulate StAR via cAMP-independent pathways. These include epidermal growth factor (EGF), macrophage-derived factors such as IL-1 and TNFα, hormones such as prolactin and gonadotropin-releasing hormone (GnRH), chloride ions, and calcium messenger systems. In addition, a protein kinase C pathway can be activated resulting in increased transcription and translation of StAR (Stocco et al., 2005; Manna et al., 2007). Leutinizing hormone binding to its receptor can release arachidonic acid (AA) and metabolites of AA can regulate StAR gene expression (Wang et al., 1999).

Pathology

The mammalian testis is composed of a fibrous tunic that surrounds convoluted seminiferous tubules and supporting stroma. In a microscopic section the tubules make up the majority of the cellular structure present and contain mostly gametes in various stages of differentiation from spermatagonia to spermatozoa in addition to supportive cells, such as Sertoli cells. Surrounding the tubules is a small amount of connective tissue that contains blood vessels, nerves, and interstitial cells of Leydig (Figure 109.2). Normal LCs have a wide variation in morphology. They can be spindle shaped with little cytoplasm but are typically round and contain large amounts of lightly eosinophilic cytoplasm. In humans and the wild bush rat, the LC cytoplasm contains elongated, cigar-shaped structures known as crystals of Reinke. These crystals can be seen with light microscopy and are found only in adults. Their origin and function are unknown (Young and Heath, 2000).

Human Relevance Of Rodent Leydig Cell Tumors-1

FIGURE 109.2 There are low numbers of Leydig cells in the interstitium surrounding seminiferous tubules of this normal rat testis (H&E, 200×).

In rodents, hyperplasia of LCs is characterized by aggregates of cells that focally or diffusely expand the interstitium between seminiferous tubules. Cells are typically round with centrally located nuclei and granular, eosinophilic cytoplasm. Occasionally, spindle cells with little cytoplasm and dark nuclei are also found (Boorman et al., 1990).

Leydig cell adenoma begins as hyperplasia and consists of one or more foci of expansile LCs that typically compress adjacent tubules. The distinction between hyperplasia and adenoma is difficult as cellular characteristics cannot distinguish between them. Most often size is used as a morphologic criteria to differentiate hyperplasia from adenoma. One recommendation from the National Toxicologic Program (NTP) is to use the size of adjacent tubules as a threshold. When the lesion grows larger than the diameter of the adjacent tubule, it is diagnosed as adenoma. In 2005 toxicologic pathology societies from around the world began an initiative to standardize nomenclature for proliferative and nonproliferative lesions in rats and mice. This initiative is termed the International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice (INHAND) (Mann et al., 2012). The INHAND focuses on specific organ systems. For the male reproductive system,