Hormones of the Testes

C H A P T E R

24 Hormones of the Testes Eleonora Zakharian Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine, Peoria, IL, United States

1. STEROID HORMONES Androgens are the main steroid hormones synthesized by the testis. Androgens and in particular testosterone are produced by the interstitial Leydig cells. The pituitary gonadotropin luteinizing hormone (LH) is the key factor that regulates testicular steroidogenesis (Huhtaniemi et al., 2011a). Androgens are essential for typical masculine functions of the body, including sexual differentiation, development of secondary sex characteristics, spermatogenesis, the masculine features of the musclebone system, and sexual behavior (Eberhard Nieschlag and Nieschlag, 2012). The downstream signaling apparatus of the prevalent androgendtestosteroned presents a complex mechanism and involves genomic and nongenomic pathways. The genomic signaling pathway is achieved when androgens bind and activate their cognate nuclear receptor and transcription factor, the androgen receptor (AR) protein, while a nongenomic/rapid signaling mechanism involves an ionotropic testosterone receptor residing at the plasma membrane, the transient receptor potential melastatin 8 (TRPM8) protein. Both pathways are discussed subsequently.

2. TESTICULAR STEROIDOGENESIS The Leydig cells located in the testicular interstitium present the major production site for androgens. Leydig cells produce about 95% of circulating testosterone under the tight control of the gonadotropin LH, the LH receptor expressed on the plasma membrane, and their downstream signaling cascade. The remaining androgens are produced from the adrenal steroidogenesis with a little contribution from peripheral tissues. The Leydig cells contain all the enzymes required for the synthesis of testosterone and its metabolites: Hormonal Signaling in Biology and Medicine https://doi.org/10.1016/B978-0-12-813814-4.00024-9

dihydrotestosterone (DHT) or estradiol (Payne, 2007). In the adult testis, Leydig cells develop from peritubular and perivascular mesenchymal-like cells, and their differentiation into mature Leydig cells is controlled by LH, numerous growth factors, and differentiation factors derived from Sertoli cells (Lei et al., 2001; Sriraman et al., 2005). The key precursor to all steroidal hormones is cholesterol. Leydig cells producing androgens have several sources of cholesterol, including (1) produced from acetyl-coenzyme A (acetyl-CoA), (2) derived from cholesterol esters stores, (3) supplied cholesterol from lipoprotein, and (4) plasma membrane-derived cholesterol sequestered upon hormonal stimulation. The most abundant sources of cholesterol are low- and high-density lipoprotein cholesterol complexes delivered by the receptormediated endocytosis. Inside of cells, cholesterol is esterified and stored in the lipid droplets until its utilization (Azhar and Payne, 2007). In the next step, cholesterol is translocated from lipid droplets to the outer membrane of mitochondria. Upon the hormonal stimulus from gonadotropins, cholesterol is further transferred from the outer to the inner mitochondrial membrane. Two regulating proteins play a critical role during this transfer step of cholesterol. They include the steroidogenic acute regulatory protein (StAR) (Clark and Stocco, 1996; Miller, 2007), and the peripheral-type benzodiazepine receptor (Papadopoulos et al., 2007). The StAR protein is expressed in gonadal and adrenal cells in response to stimuli for steroidogenesis, including LH. StAR plays a critical role in steroidogenesis, as its functional knockdown leads to the development of congenital lipoid adrenal hyperplasia (CAH) and dramatic suppression of steroidogenesis (Bose et al., 1996). Furthermore, patients with a mutation inactivating StAR gene are unable to produce sufficient amounts of steroids and develop a life-threatening form of CAH (King et al., 2011).

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At the inner mitochondrial membrane, cholesterol undergoes the first step of steroidogenesis by conversion to pregnenolone. This step is catalyzed by the cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc, or CYP11A1) and auxiliary electron transferring protein, which adjourns the mitochondrial phase (Fig. 24.1). For processing of the subsequent steps, pregnenolone is then transported to the smooth endoplasmic reticulum, where it further undergoes enzymatic reactions of two divergent pathways, D5 or D4. The microsomal enzyme 3b-hydroxysteroid dehydrogenase/ D5/D4 isomerase (3bHSD) catalyzes both conversions of the hydroxyl group to a keto group on carbon 3 and the isomerization of the double bond from the B ring (D5 steroids) to the A ring (D4 steroids) (Thomas et al., 1989; Lorence et al., 1990). In the D4 pathway, 3b-HSD catalyzes the conversion of pregnenolone to progesterone. These signaling pathways depend on age and species, with D5 being a dominant pathway taking place in human testis (Payne and Hales, 2004; Leinonen et al., 1981). Further, pregnenolone undergoes 17a-hydroxylation-enzymatic step to form 17a-hydroxypregnenolone and then a lyase step to produce dehydroepiandrosterone (DHEA). Both enzymatic reactions are catalyzed by cytochrome P450c17 (CYP17A1, 17a-hydrolase/ 17,20-lyase) (Fig. 24.1). As a result, 2 carbons are removed by the CYP17A1 to yield C19 steroids. Next, the 3b-hydroxy-5-ene structure of dehydroepiandrosterone is converted to the 3-keto-4-ene structure of androstenedione performed by the enzyme 3b-HSD. Alternatively, dehydroepiandrosterone can be converted to 5-androstene-3b, 17b-diol through the reduction by 17b-hydroxysteroid dehydrogenase (17b-HSD, mainly type III). In the final step, the C17-keto group of androstenedione is reduced by 17b-HSD to yield testosterone. The detailed overview of the enzymes participating in the steroidogenesis and their deficiencies can be found in the work by Miller and Auchus (Miller and Auchus, 2011). Men daily produce on average 6e7 mg of testosterone, with about 95% of this amount to be produced by the testes and the remainder derived from the peripheral adrenal androgen synthesis. Some of the testicular steroid is stored in the testis and includes intermediate products of D5 or D4 pathways. The testis secretes some of these hormones in the sulfated form. It has initially been considered that sulfated steroids have no hormonal function and present storage or secretory forms of steroids. However, recent studies provided insights into various activities of sulfated steroids. For instance, the neurosteroid pregnenolone sulfate was shown to act on different molecular targets, including modulation of activity of the N-methyl-D-aspartate (NMDA) receptors (Wu et al., 1991; Jang et al., 2004) impacting neuroplasticity

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FIGURE 24.1 Steroidogenesis pathways in the testis executed by the following enzymes to produce testosterone: the cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc, or CYP11A1); cytochrome P450c17a-hydrolase/17,20-lyase (CYP17A1); 3b-hydroxysteroid dehydrogenase/D5/D4 isomerase (3b-HSD); and 17b-hydroxysteroid dehydrogenase (17b-HSD). Testosterone is further metabolized to DHT by 5a-reductase (5a-R) or estradiol by aromatase (Aro).

(Mameli et al., 2005) or potentiation of the transient receptor potential melastatin 3 (TRPM3) ion channel, thus causing a rapid ionotropic effect (Wagner et al., 2008). Pregnenolone sulfate was shown to exert a direct action on TRPM3 channels (Uchida et al., 2016; Demirkhanyan et al., 2016), and importantly, the interaction of the steroid with the channel is mediated by the presence of the negatively charged sulfate group, while the potency and efficacy of unsulfated pregnenolone are relatively low (Wagner et al., 2008; Drews et al., 2014). The physiologic implication of this ion channel-driven

4. TESTOSTERONE IS A DIRECT PRECURSOR FOR OTHER BIOACTIVE STEROIDS

mechanism might play a role in insulin secretion and management of pronociceptive actions of TRPM3. The downstream products of testosterone can also be synthesized by the testis but in low amounts. This includes 5a-dihydrotestosterone (DHT) produced by 5a-reductase. For a long time, DHT was considered the most potent androgen due to the activation of the AR signaling pathway; however, a recently identified rapid testosterone signaling component offered alternative androgen signaling mechanisms (see later). Testosterone also can be aromatized by CYP19 (P450arom) to form estrogens.

3. SECRETION AND TRANSPORT OF TESTICULAR STEROIDS Steroids are lipid-soluble molecules and passively migrate through the membrane of Leydig cells to circulation. The most abundant steroids comprise testosterone and a sulfated form of pregnenolone, dehydroepiandrosterone, and 5-androstene-3b, 17b-diol. Bioactive steroids are not stored within the testes, unlike their sulfate conjugates. The rhythm of testosterone production is relatively high, with about 200 reactions to produce the 6e7 mg daily.

3.1 Associating Proteins In circulation, about 1%e2% of testosterone appears in free or unbound to protein form, 44% is bound to the sex hormone-binding globulin (SHBG), while 54% to albumin and other proteins (Bhasin et al., 2007). SHBG is a b-globulin with nonidentical subunits that contain the androgen binding sites. The androgen binding proteins essentially affect the availability of free steroids, thus playing a critical role in regulating androgen action (Laurent et al., 2016). Even though albumin binds steroids with low affinity (micromolar range), it essentially impacts steroid availability due to its high abundance (Dunn and Rodbard, 1981). In contrast, SHBG binds androgens, estrogens, and their precursors and metabolites (Dunn and Rodbard, 1981; Avvakumov et al., 2010; Cherkasov et al., 2008; Grishkovskaya et al., 2002) with high specificity and affinity (nanomolar range) (reviewed in Hammond, 2016; Schiffer et al., 2018). The free fraction of steroid and bioactive fraction, which includes unbound and albumin-bound steroid, can be estimated using total androgen, albumin, and SHBG concentrations. The complex of testosterone and SHBG is not permeable to cells, and release of the steroids from the protein mostly takes place in the

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capillaries, where the interaction of SHBG with the endothelial glycocalyx reduces the protein affinity to steroids (Huhtaniemi et al., 2011b). On the contrary, testosterone is easily dissociable from albumin, and therefore the albumin-bound form of testosterone belongs to the active fraction of androgens (Hammond, 2016; Huhtaniemi et al., 2011b).

4. TESTOSTERONE IS A DIRECT PRECURSOR FOR OTHER BIOACTIVE STEROIDS: DHT AND ESTROGEN The importance of testosterone is also underlined by the fact that this prevalent male hormone, besides being a source of its reduced analog DHT, also is a direct precursor for the prevalent female hormone: estradiol. These two divergent enzymatic reactions are executed by the enzymes 5a-reductase, which catalyzes the conversion of testosterone to DHT, and aromatase, which aromatases testosterone to estradiol (Fig. 24.1). Due to its higher affinity to the classical AR, DHT (w10 time more potent than testosterone) has been considered a more potent bioactive androgen than testosterone itself. However, a recent discovery of a direct ionotropic testosterone receptor, TRPM8, that resides on the plasma membrane and possesses 1000-fold higher sensitivity to testosterone over DHT (ranging in low picomolar concentrations) essentially reinforced the physiologic role of testosterone (discussed later). Along with other androgens, estradiol also has a role in regulating other testicular steroids, and testis can secrete both DHT and estradiol in small amounts. Circulating DHT is primarily formed in the various androgen target organs, including hair follicles and the prostate. In contrast, estradiol is mainly formed in the adipose tissue. The 5a-reductase enzyme exists in two isoforms, type 1 and 2 (Mahendroo and Russell, 1999). Type 1 is expressed in the sebaceous gland and the liver, while type 2 is present in the male urogenital tract, genital skin, and liver. Androgens are known to upregulate 5a-reductase in the prostate. Thyroid hormones regulate it in the liver and insulin-like growth factor 1 (IGF-1) in the skin fibroblasts. Estradiol present in male circulation is primarily derived from the adipose tissue, and 25% of it is secreted by the testes (Bhasin et al., 2007). Estrogens play essential roles in males. Insufficient estrogen function in males can lead to epiphyseal closure, osteoporosis, insulin resistance, and abnormalities in plasma lipids.

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5. METABOLISM OF TESTICULAR STEROIDS One of the primary mechanisms for inactivation of reactive androgens is an alteration of their solubility. During this process, androgens that are naturally highly hydrophobic attain modifications that add hydrophilic content, thus impacting the ability of the steroid to penetrate the membrane (Payne and Hales, 2004; Bhasin et al., 2007; Sundaram et al., 1996). These modifications include conjugation with sulfates and glucuronides. The catabolic reactions of a reductive nature are mainly occurring in the liver. The liver controls the activity and clearance of steroid hormones by catalyzing the extensive phase 1 and 2 metabolisms. The hepatic phase 1 metabolism of androgens undergoes via the following reactions: (1) 5a/b-reduction of the D4double bond followed by 3a/b-reduction of the 3-ketone; (2) oxidations by a large set of hepatic P450 enzymes; (3) hydroxysteroid dehydrogenase-induced reactions of 11b- and 17b-hydroxyls and hydroxyls introduced by P450s. The liver contains high levels of 5b-reductase (AKR1D1), which leads to the formation of 5-b-reduced androgens. In contrast, peripheral tissues primarily produce 5a-reduced metabolites (Charbonneau and The, 2001; Chen and Penning, 2014; Penning, 2010). A formation of the 3a-reduced counterpart of a 5b-reduced steroid leads to the formation of 3aOH-5b-reduced products (Jin et al., 2011). Hydroxylation is catalyzed by several P450s, including the CYP1, CYP2, CYP3, and CYP7 families, some of which also can induce additional oxidation of a hydroxyl group to its ketone (Niwa et al., 2015). CYP3A4 is the most abundant P450 in the liver (Shimada et al., 1994), and it predominantly catalyzes the 6b-hydroxylation of testosterone and 16a- and 7a-hydroxylation of DHEA (Niwa et al., 2015). Hepatic phase-2 metabolism promotes sulfation and glucuronidation. The enzymes that catalyze the addition of sulfate groups, sulfotransferase (SULT), are highly expressed in the liver, with SULT2A1 and SULT2B1 isoforms involved in androgen metabolism (Falany, 2001; Riches et al., 2009). A hepatic activity of androgen metabolism is not only prompt for sulfation but also for glucuronidation. The action of uridine diphosphateglucuronosyl transferase-induced androgen glucuronidation is prevalent in the liver compared to other tissues (Belanger et al., 2003; Rittmaster et al., 1993). Conjugated metabolites are further released by hepatic cells into circulation via an active transport. Although the liver is the primary site of action for androgen utilization, their metabolism can also undergo in several different tissues (for review, see Schiffer et al., 2018). About 90%

of the androgen metabolites are excreted with urine and 10% with feces. Urinary androgen metabolites contain 20%e40% in the form of glucuronides, 40% sulfates, and the rest in the free form (Huhtaniemi et al., 2011b). Importantly, the levels of produced androgen metabolites in women are comparable to that of men (Labrie et al., 1997; Trabert et al., 2016; Zang et al., 2017).

6. FUNCTION: TESTOSTERONE AND DHT RECEPTORS: CROSSTALK OF GENOMIC AND NONGENOMIC MECHANISMS Androgens regulate growth and development by activating genomic receptor pathways. Genomic actions of androgens are mediated by the AR protein (gene name NR3C4), which is a member of the nuclear receptor family of transcription factors (Beato et al., 1996; Bruck et al., 2005). A steroid-unbound form of AR is predominantly present in the cytoplasm where it interacts with various proteins, including heat-shock proteins. Upon androgen binding to AR, the receptor undergoes dimerization, followed by the steroid-AR complex translocation to the nucleus, where it interacts with various target promoters and stimulates gene expression (Wierman, 2007). Within the gene promoter of the target genes, AR binds to the specific region called androgen response elements (AREs) (Fig. 24.2). Androgens can also exert rapid/nongenomic actions evidenced in different cellular systems (Wehling, 1997; Revelli et al., 1998). These actions do not involve the canonical AR pathway; rather, they suggest participation of a signal-generating membrane surface receptor. In particular, rapid testosterone-induced intracellular Ca2þ elevations have been observed in a variety of cell types, including rat Sertoli cells, human prostate cells (LNCaP and PC3) (Lyng et al., 2000), rat heart myocytes (Koenig et al., 1989), male (but not female) rat osteoblasts (Lieberherr and Grosse, 1994), and mouse T cells lacking the functional AR protein (Benten et al., 1997, 1999). Recently, a missing element in rapid testosterone signaling the TRPM8 protein emerged as a highly potent ionotropic testosterone receptor (Asuthkar et al., 2015a, b, c, 2016). Initially, TRPM8 was recognized as a major receptor for a wide range of cold temperatures in the peripheral nervous system (Bautista et al., 2007; Dhaka et al., 2007; Colburn et al., 2007), where the channel is also responsible for sensing chemical compounds such as menthol, icilin, eucalyptol, geraniol, and linalool (Behrendt et al., 2004; McKemy et al., 2002; Peier et al., 2002). Apart from the nervous system, the TRPM8 protein is expressed in various tissues, including the lungs,

7. TESTICULAR PROTEIN AND PEPTIDE HORMONES: INHIBIN, ACTIVIN, AND FOLLISTATIN

heart, and prostate (Sabnis et al., 2008; Tsavaler et al., 2001). Although the role of TRPM8 as the cold and menthol receptor has been well established in the peripheral nervous system (Bautista et al., 2007; McKemy et al., 2002; Peier et al., 2002), its role in other tissues was not well understood since no endogenous agonists of TRPM8 were previously identified. Later, biochemical and biophysical studies demonstrated a direct link of TRPM8 and testosterone (Asuthkar et al., 2015a, b). Immunohistochemistry and coimmunoprecipitation experiments with human prostate tissues and cultured cells revealed that endogenous testosterone directly interacts with TRPM8. Further, it was demonstrated that testosterone directly activates TRPM8 in different cellular systems by eliciting a rapid Ca2þ influx. Specifically, testosterone-induced TRPM8 responses were detected in prostate cancer cells (PC3), dorsal root ganglion neurons, hippocampal neurons, and human embryonic kidney (HEK-293) cells. Besides, testosterone exerted its agonist action on purified TRPM8 channels incorporated into planar lipid bilayers. The addition of testosterone in picomolar concentrations resulted in the full opening of TRPM8 leading to ionic current (Fig. 24.2).

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Despite that testosterone regulates many physiological processes, its direct role was mainly unknown. Indeed, the most recognized action of testosterone in anabolic processes is mediated by its reduced analog DHT, to which testosterone is converted by 5a-reductase when it enters the cell. It is, in fact, DHT that binds AR and stimulates protein synthesis by transactivation of nuclear genes. Alternatively, testosterone is a direct precursor of aromatase-mediated conversion to estrogen, which then activates the estrogen receptors (ERs) and their subsequent targets (Fig. 24.2). Remarkably, TRPM8 and AR have reverse preferences for binding testosterone and DHT: DHT has nearly a 10-fold higher affinity (100 pM) for the AR than does testosterone (1 nM) (Litvinov et al., 2003), while testosterone has almost 1000-fold higher potency and specificity (22 pM) for TRPM8 than does DHT (23 nM) (Asuthkar et al., 2015b). Finally, residing on the plasma membrane, TRPM8 engages in direct contact with testosterone before it enters the cell and is further converted to DHT. Hence, TRPM8 stands as the first immediate receptor for testosterone with all its physiologic implications.

7. TESTICULAR PROTEIN AND PEPTIDE HORMONES: INHIBIN, ACTIVIN, AND FOLLISTATIN

Testosterone in rapid and genomic signaling ERE

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FIGURE 24.2 A molecular model of testosterone-evoked genomic and nongenomic signaling: The model depicts testosterone-induced TRPM8 activity that results in rapid Ca2þ or Naþ influx. Phosphatidylinositol 4,5-bisphosphate (PIP2) regulates this activity as the prime TRPM8 activity cofactor. 5a-reductase (5a-R) converts testosterone (T) to dihydrotestosterone (DHT). Aromatase (Aro) converts testosterone (T) to estradiol (E). DHT can bind to the transcription factor androgen receptor (AR), with subsequent activation of the target genes at the androgen receptor elements (ARE). Estradiol (E) can bind to its cognate receptors, estrogen receptors (ERs), thus regulating their target genes via interacting with the estrogen receptor elements (ERE).

Testis also synthesizes and secretes a few regulatory protein hormones. The first member of these hormones named inhibin was described in 1932 (McCullagh, 1932) and cloned and characterized in 1985 (Ying, 1988; de Kretser and Robertson, 1989). Inhibin is a gonadal glycoprotein that regulates FSH synthesis and release. Inhibin forms heterodimers of 32 kDa linked by disulfide bonds composed of an a-subunit and one of two b-subunits (bA and bB). Both forms of dimers, inhibin-A (a-bA) and inhibin-B (a-bB), suppress the FSH release. It is interesting that the formation of homodimers instead of heterodimers results in the assembly of a different protein, activin, that possesses an opposite function and is known to stimulate synthesis and release of FSH from the pituitary. Activins can form b-b-dimers of which the bA-bA and bB-bB homodimers and bA-bB heterodimers were shown to stimulate FSH secretion from the pituitary. Thus, combinatorial assembly of different subunits results in multiple hormonal activities regulating the hypothalamic-pituitary-gonadal axis. The mRNA encoding inhibin and activin are most abundant in the ovary and the testis, but they are also present in other tissues (Meunier et al., 1988).

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Apart from their endocrine role, inhibin and activin were shown to regulate the growth and differentiation of various cell types, including anterior pituitary cells, gonadal and neuronal cell lines, and hematopoietic progenitor and erythroleukemia cells (Meunier et al., 1988). The interacting proteins for inhibin and activin include follistatin and a-2 macroglobulin. Follistatin was identified for its ability to suppress pituitary FSH release, and it was found to directly interact with activin (Nakamura et al., 1990) and inhibin, although to a lesser extent (Shimonaka et al., 1991; Krummen et al., 1993). Activin binds to two types of receptors, I and II, which are the serine/threonine kinases. Inhibin can interfere with binding of activin to these receptors, and this antagonism is further enhanced by betaglycan, a membrane-bound form of peptidoglycan that acts as inhibin coreceptor. All subunits of inhibin are detected in fetal testis, although the expression pattern and prevalence to the specific cell types changes upon maturation. In the adult testis, inhibin is mainly synthesized by Sertoli cells with small quantities produced in the Leydig cells (Roberts et al., 1989; Maddocks and Sharpe, 1989). It is interesting that Sertoli cells primarily express a and bB mRNA, and weakly express bA mRNA. The Leydig cells, in contrast, predominantly express bA, very little a, and no bB mRNA. Thus, inhibin-B is the main secretary form of inhibin produced in males. Follistatin is synthesized by both Sertoli and germ cells. Regulation of inhibin synthesis is maintained by FSH, which stimulates inhibin subunit a mRNA expression in Sertoli cells but has no effect on the b subunits. Thereby, FSH secretion and inhibin levels are inversely correlated, similar to the negative feedback from testosterone, which together with inhibin provokes regulation of FSH synthesis and release (de Kretser et al., 2004; Luisi et al., 2005). On the other hand, the synthesis of activin subunits bA and bB is independent of FHS. Type I and II activin receptors are present in Sertoli cells, spermatogonia, and some spermatocytes. These cell types, therefore, are the target for activin action, while Leydig cells are confined target for inhibin action (Bernard and Woodruff, 2002).

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