ANDROLOGY
ISSN: 2047-2919
REVIEW ARTICLE
Correspondence: Brian Le, The James Buchanan Brady Urological Institute, Johns Hopkins Hospital, 600 North Wolfe Street, Marburg 145 Baltimore, MD 21287-2101, USA. E-mail: [email protected]
Keywords: ageing, androgens, Leydig cells, LH, LH receptor, testosterone
New targets for increasing endogenous testosterone production: clinical implications and review of the literature 1
B. Le, 2H. Chen, 2B. Zirkin and 1A. Burnett
1
Received: 3-Dec-2013 Revised: 3-Mar-2014 Accepted: 6-Apr-2014
The James Buchanan Brady Urological Institute, Johns Hopkins Hospital, Baltimore, MD, USA, and Department of Biochemistry and Molecular Biology, The Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, USA 2
doi: 10.1111/j.2047-2927.2014.00225.x
SUMMARY Over the past several decades, our understanding of the regulatory mechanisms of testosterone production has increased significantly. Concurrently, the medical treatment of hypogonadism, particularly in the ageing male has increased. This review article consolidates some of our insights into the regulatory mechanisms of endogenous testosterone production and examines promising new targets that may allow endogenous production of testosterone to be re-established in males with primary hypogonadism. We examined the published scientific literature regarding regulatory mechanisms of testosterone biosynthesis with a focus on Leydig cell physiology and small-molecule regulation that resulted in increased testosterone production. We identified several pathways that have been manipulated pharmacologically to increase Leydig cell testosterone production, including phosphodiesterases, the cholesterol translocator protein, the electron transport chain of mitochondria, cyclooxygenases and osteocalcin. Manipulation of these pathways with small molecules has helped further our understanding of the regulatory pathways of testosterone biosynthesis. Herein, we identified five future targets that might promote increased endogenous testosterone production through the Leydig cell instead of relying on exogenous testosterone administration.
INTRODUCTION In recent years, public awareness of the signs and symptoms of low serum testosterone or hypogonadism has increased. Although they are often vague, these include common symptoms of fatigue, worsening cognition, decreased libido, decreased muscle mass, depressed mood and sexual dysfunction (Bassil, 2011). Low serum testosterone, the end result of decreased endogenous production and bioavailability of testosterone, is believed to affect ~5 million American men (Araujo et al., 2007). Clinically, physicians see two main presentations for hypogonadism: (i) the younger male presenting with infertility or subfertility, and (ii) the older male with signs and symptoms of low testosterone who is seeking evaluation. In the first scenario, a history, physical examination and a panel of tests (including semen analysis and serum levels of FSH, LH, testosterone, estradiol and prolactin) are performed to determine whether hypogonadism is associated with, and perhaps the cause of the male infertility, as opposed to other causes such as obstruction or genetic abnormalities. If low intratesticular testosterone (ITT) is a suspected aetiology, which would be © 2014 American Society of Andrology and European Academy of Andrology
suggested by low serum testosterone levels, LH levels are examined to help differentiate between hypogonadotropic hypogonadism (low LH, low T) vs. primary hypogonadism (high LH, low T). Hypogonadotropic hypogonadism is the rarer of the two entities and is associated with Kallman syndrome, idiopathic hypogonadotropic hypogonadism and congenital adrenal hyperplasia (Duke et al., 1995; Layman et al., 2002; Trussell, 2013). In the case of hypogonadotropic hypogonadism, treatment is directed towards stimulation of the Leydig cell through the LH receptor by pharmacologic means [recombinant LH, hCG, pulsatile gonadotropin releasing hormone (GnRH), or clomiphene], the objective is to correct putative low intratesticular testosterone levels and thus restore spermatogenesis (Hamada et al., 2012). Primary hypogonadism, the more common entity, can be more challenging to manage, but some may still respond to LH receptor stimulation (Hussein et al., 2005; Trussell, 2013; Hotaling & Patel, 2014). Hypogonadism has been shown to be associated with nonobstructive azoospermia in as many as 47% of patients undergoing testicular sperm extraction, and therefore means by which Andrology, 1–7
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to modulate testosterone levels have been areas of extreme interest (Ramasamy et al., 2012). Unfortunately, treatment with exogenous LH/hCG/clomiphene in individuals with primary hypogonadism is often ineffective in stimulating Leydig cell testosterone production to raise serum and intratesticular testosterone levels (Bremner et al., 1994; Zwart et al., 1996; Hussein et al., 2005; Reifsnyder et al., 2012; Trussell, 2013). Moreover, the exogenous administration of testosterone itself, although it increases serum testosterone levels, is unlikely to increase intratesticular testosterone levels. Rather, such treatment is likely to reduce these levels by negative feedback inhibition of pituitary LH secretion (Kamischke & Nieschlag, 1999). This presents a conundrum for the clinician who seeks to increase intratesticular testosterone, but has few options that do not act through the LH receptor. In the second scenario, that of the older hypogonadal male, he also suffers from the problem of decreased Leydig cell responsiveness to LH stimulation and low endogenous testosterone production, and is thus a form of primary hypogonadism. Primary hypogonadism is the predominant type found in this population (Sih et al., 1997; Harman et al., 2001). In men, serum testosterone levels typically begin to decline in the fifth decade of life and are usually accompanied by increasing serum levels of FSH and stable or increased levels of LH (Zirkin & Chen, 2000; Midzak et al., 2009). However, in this situation, the men are usually less concerned about fertility and so they routinely turn to exogenous administration of testosterone. This is by far the larger target market for pharmaceutical companies, and over the past several years, there has been increased marketing by such companies. Indeed, advertising expenditures on exogenous testosterone supplementation have increased 1000% from 2010 to 2012 (Donohue et al., 2007). Correction of testosterone levels of less than 300 ng/dL in symptomatic men often leads to symptomatic improvement and improved quality of life (Sih et al., 1997; Gruenewald & Matsumoto, 2003). Furthermore, developments in delivery systems including gels, roll-on applications and subcutaneous pellets, all of which bypass first pass metabolism in the liver, have made testosterone replacement increasingly accessible and palatable to men. However, exogenous testosterone replacement is not without its risks and drawbacks, including increased risk of cardiovascular events, prostate growth, worsening sleep apnoea, testicular atrophy, infertility and exacerbation of certain types of cancers (Corona et al., 2013). Additionally, the use of exogenous testosterone ultimately leads to LH down-regulation and shutdown of endogenous production of testosterone and thus reductions in intratesticular testosterone levels that are essential for spermatogenesis (Zirkin et al., 1989). The long-term consequences of exogenous testosterone supplementation on the intratesticular environment are not entirely clear. This review article examines promising new targets that may allow endogenous production of testosterone to be reestablished in males with primary hypogonadism, and thus increase intratesticular testosterone levels. The objectives of such approaches should be to: (i) maintain an intratesticular testosterone environment that allows/promotes normal spermatogenesis, (ii) avoid side effects of exogenous testosterone supplementation and (iii) maintain the negative feedback mechanism of LH that prevents testosterone levels from becoming supratherapeutic. We review new potential treatments that have 2
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ANDROLOGY these advantages, and discuss the clinical promise of such findings.
INTRATESTICULAR TESTOSTERONE LEVELS The focus on intratesticular testosterone levels as opposed to serum testosterone levels is an important distinction. It is well established that the total testosterone concentration within the testes is far higher than that in the serum, with intratesticular testosterone levels 8- to 30-fold higher in rodents and 20- to 100-fold higher in humans (Jarow & Zirkin, 2005). However, the relationship between serum testosterone levels and intratesticular testosterone levels is non-linear and has considerable variability. For example, Jarow et al., using percutaneous aspiration of the testes in fertile men in a clinical setting, determined that the mean total testosterone concentration within the testes was 600 ng/mL, compared to 5 ng/mL in serum, but with considerable variability between individuals (Jarow et al., 2001). The aetiology of this concentration differential is likely because of local testosterone production by Leydig cells and the countercurrent mechanism in the pampiniform plexus (Jarow & Zirkin, 2005). When clinically low serum testosterone levels are detected, it may be the end result of the disruption of steroidogenic production by Leydig cells, although other explanations are possible. Testosterone mediates its effects on spermatogenesis by binding to the androgen receptor found on Sertoli cells, and also binds to receptors in Leydig and peritubular cells (Bremner et al., 1994; Page, 2011). Under normal conditions, high intratesticular concentrations of testosterone are a requirement for the maintenance of normal spermatogenesis and thus the prevention of testicular atrophy. For example, in the rat reductions of intratesticular testosterone levels to 20% of normal values do not seem to affect spermatogenesis, but reductions below this level result in dramatic decreases in sperm production. However, 20% of normal intratesticular testosterone represents a far higher level (4- to 5-fold) than normal serum levels (Zirkin et al., 1989). Data such as these suggest an association between hypogonadism and male infertility in young men that may be treated with correction of intratesticular testosterone levels. However, the exact relationship between endocrine dysfunction, intratesticular testosterone levels and infertility are poorly understood. This is further complicated as male infertility is a heterogenous entity with presentations from azoospermia to subfertility with normal semen parameters. Indeed only 3% of male infertility cases have an endocrine aetiology, as measured by serum hormone values, and identified as a treatable cause of infertility, despite 30–70% of infertile men having some degree of endocrine dysfunction (Hotaling & Patel, 2014). There are very limited data on ITT in the setting of male infertility from humans. In many cases, ITT levels may be normal or increased in patients with non-obstructive azoospermia at the time of testicular sperm extraction which is believed to be owing to a primary genetic defect with endocrine compensation (Shinjo et al., 2013).
PATHOPHYSIOLOGY OF DECREASED TESTOSTERONE PRODUCTION IN THE AGEING MALE Cross-sectional and longitudinal ageing studies have demonstrated that ageing is associated with a progressive decline in serum testosterone levels (Harman et al., 2001). The prevalence of clinically symptomatic low testosterone is estimated to be © 2014 American Society of Andrology and European Academy of Andrology
TARGETS FOR ENDOGENOUS TESTOSTERONE PRODUCTION
20% of men at age 60, and 50% at age 80 (Gruenewald & Matsumoto, 2003). Despite these decreases in serum testosterone, LH levels are usually unchanged or increased, while FSH levels are usually increased compared to younger males, suggesting a primary testicular deficiency (Zwart et al., 1996). Although agerelated changes in GnRH gene expression and the amplitude of LH pulses have been shown as well, this is believed to be less important than decreased Leydig cell responsiveness of LH, which has been demonstrated in rats (Bonavera et al., 1997, 1998) as well as in humans (Liu et al., 2005a,b). In a study conducted by Liu et al., they administered recombinant human LH in a pulsatile fashion to healthy young and old men and examined the steroidogenic response. They noted a blunted testosterone peak, amplitude and response to stimulation compared to younger men (Liu et al., 2005a,b). Studies examining the differences between young and aged Leydig cells that have come primarily from the study of rat models of ageing have elucidated several key differences including: (i) decreased ability to synthesize and mobilize cholesterol for steroidogenesis by aged Leydig cells (ii) decreased number of LH receptors, (iii) decreased activity and expression of cholesterol side-chain cleavage enzyme (CYP11A1) and (iv) decreased activity of steroidogenic enzymes in the smooth endoplasmic reticulum involved in converting pregnenolone to testosterone (Zirkin & Chen, 2000).
CURRENT APPROACHES Current approaches to the treatment of male hypogonadism begin with a careful clinical history and physical examination to identify agents or aetiologies that may be adversely affecting steroidogenesis and spermatogenesis, such as anabolic steroid use, medications, presence of varicocoeles and trauma. When possible, these conditions are treated. The aetiology may be identified as a distal (pituitary/hypothalamus/exogenous) or a local (testicular) deficiency, termed secondary and primary hypogonadism, respectively. In these cases, clinicians turn to pharmacologic means to stimulate testosterone production, or to provide testosterone via exogenous administration so as to counter the systemic effects of hypogonadism. Pharmacologic attempts at enhancing endogenous production of testosterone have been at best modestly effective, and only in select conditions, notably clearly identifiable deficiencies in central stimulation of otherwise normal testes (Bobjer et al., 2012). One current approach is to manipulate the hypothalamic–pituitary–gonadal (HPG) axis. Under normal conditions, the hypothalamus releases gonadotropin releasing hormone (GnRH) which stimulates the anterior pituitary to release FSH and LH in a pulsatile fashion (Conn & Crowley, 1991). LH passes through the circulation and binds to the LH receptor located on Leydig cells to stimulate production of testosterone; FSH binds to Sertoli cells which support spermatogenesis. For aetiologies of male infertility, such as in Kallman’s syndrome or hypogonadotropic hypogonadism, deficiencies in LH production can be overcome by pharmacologic activation of LH or the LH receptor (Hamada et al., 2012). There are two main ways this is accomplished: direct stimulation of testosterone production by administering LH or hCG, and indirect effects on testosterone production with clomiphene or aromatase inhibitors (Page, 2011). With direct stimulation, recombinant LH is injected into the bloodstream and binds directly to the Leydig cell LH receptor to stimulate the Leydig cells to produce testosterone. With © 2014 American Society of Andrology and European Academy of Andrology
ANDROLOGY exogenous administration of hCG, the common alpha-subunit is homologous to LH, while the beta-subunit has considerable similarity but distinguishes hCG from LH (Pierce & Parsons, 1981). When administered by injection, hCG also directly stimulates the LH receptor, and promotes testosterone production. The main difference between LH and hCG is their half-lives, with LH having a circulating half-life of 25–30 min compared to hCG with a half-life of 37 h. This difference is the reason that hCG is typically used clinically for hypogonadism (Katz et al., 2012) rather than recombinant LH (Cole, 2010). With indirect stimulation, the pharmacologic approach is to stimulate endogenous LH production by inhibiting negative feedback on the HPG axis. Clomiphene has been used for decades in women to regulate or induce ovulation, and has FDA approval for that purpose (Katz et al., 2012). In men, clomiphene is used ‘off-label’ to treat hypogonadism and male infertility. It acts by preventing the binding of oestrogen, which is peripherally converted from testosterone, to receptors in the hypothalamus. This inhibition of negative feedback leads to increased GnRH stimulation of the pituitary and increased LH secretion, resulting in downstream stimulation of Leydig cells (Katz et al., 2012). An alternative approach that is used is to administer selective aromatase inhibitors which block the peripheral conversion of testosterone to estradiol through the process of aromatization (Leder et al., 2004). This again inhibits the negative feedback mechanism of the HPG axis, thus promoting downstream endogenous testosterone production. Recombinant LH, hCG, clomiphene and aromatase inhibitors all act directly or indirectly through stimulation of the LH receptor located on Leydig cells. However, in patients with normal or high LH levels and persistent hypogonadism, increasing stimulation generally does not result in increased testosterone production. In fact, hypogonadism in the vast majority of patients is not because of central deficiencies (Ramasamy et al., 2012), but rather is due to decreased responsiveness of the Leydig cell to LH (Grzywacz et al., 1998; Midzak et al., 2009). Thus, methods that aim to enhance testosterone production through LH stimulation are inherently limited, and in particular have not been very effective clinically in the ageing population (Chen et al., 2002). Clearly, there is a need to examine alternative approaches to increasing intratesticular testosterone levels and endogenous production.
FUTURE TARGETS AND APPROACHES Discussions of the possibility of identifying LH-independent mechanisms by which to increase testosterone inevitably must start with an understanding of how testosterone production by Leydig cells is regulated normally, and what is right and wrong with these cells when they no longer respond well to LH. The mechanism of LH receptor stimulation of Leydig cell testosterone biosynthesis has been an area of intense study. Figure 1 highlights pathways involved in cell signalling related to testosterone biosynthesis that have small-molecule targets. There are additional regulatory mechanisms that have been simplified for clarity. Stimulation of Leydig cell testosterone biosynthesis involves the following: LH receptor stimulation leading to cAMP formation; activation of protein kinase A; phosphorylation of transcription factors GATA-4 and CREB; regulation of interaction of StAR and translocator protein (TSPO), leading to translocation Andrology, 1–7
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Figure 1 Signalling pathways involved in Leydig Cell steroidogenesis and potential targets for enhancing testosterone production (in bold). Legend: ATP, Adenosine Triphosphate; cAMP, Cyclic Adenosine Monophosphate; CBP, CREB Binding Protein; CREB, cAMP Response Element-Binding Protein; COX-2, Cyclooxygenase 2; COX2i, Cyclooxygenase 2 inhibitor; GATA-4, Transcription Factor GATA4; GPRC6A, G-protein-coupled receptor encoded by GPRC6A; hCG, Human Chorionic Gonadotropin; LH, Luteinizing Hormone; LH-R, LH Receptor; PKA, Protein Kinase A; PKAc, Protein Kinase A Catalytic subunit; SF-1, Steroidogenic Factor 1; stAR, Steroidogenic Acute Regulatory Protein; TBXAS inhibitor, Thromboxane A Synthase inhibitor; TSPO, Translocator Protein.
of cholesterol into the mitochondria (Manna et al., 2009; Papadopoulos & Miller, 2012); conversion of cholesterol to pregnenolone in the mitochondria; and translocation of pregnenolone to the smooth endoplasmic reticulum to be converted into testosterone and secreted by the Leydig cell (Fig. 1). The rate-limiting step in this pathway is the translocation of cholesterol from intracellular stores to the inner mitochondrial membrane. Two proteins that are critical to this translocation are StAR and the cholesterol TSPO (Zirkin & Chen, 2000; Culty et al., 2002). The reasons for the decline in testosterone biosynthesis under conditions of primary hypogonadism are numerous. Several key observations have been noted: decreased responsiveness of the LH receptor; decreases in the StAR protein; decreases in TSPO; increases in COX2 expression; reduced conversion of cholesterol to pregnenolone in the mitochondria; and reduced conversion of pregnenolone ultimately to testosterone in the smooth endoplasmic reticulum. Together these observations point to downstream deficiencies in signalling that converge on cholesterol translocation into the mitochondria, the rate-limiting step in testosterone formation and thus on the StAR protein and on TSPO, both integrally involved in cholesterol transfer to the inner mitochondrial membrane (Tsai & Beavo, 2011; Chung et al., 2013). Based on these understandings, the following describes several potential targets for increasing endogenous testosterone production that might be of particular clinical promise given the pharmacology, the strength of the data, and toxicity profiles. Furthermore, it might be possible to use these agents in concert to produce a cocktail to rescue deficient testosterone steroidogenesis. 4
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Phosphodiesterases LH binds to LH receptors which, in turn, couple to G proteins, stimulating adenylyl cyclase (AC) to convert ATP into cAMP. This is followed by activation of protein kinase A, which initiates transcriptional events that produce the steroidogenic proteins which, in Leydig cells, form testosterone from cholesterol (Fig. 1). The production of cAMP is of critical importance in testosterone formation. Phosphodiesterases (PDEs) play an important role in regulating the balance of cyclic nucleotides. The level of cAMP is determined by the rates of synthesis by ACs and degradation by PDEs (Tsai & Beavo, 2011). There are several subtypes of PDE. The main types involved in steroidogenesis by Leydig cells are those that mediate cAMP conversion to 5’AMP, including PDE8A, PDE4 and PDE2A. The most promising of these is PDE8A, which hydrolyzes cAMP exclusively and is found in high levels in the testes, spleen, colon, small intestine, ovary, placenta and kidney (Wang et al., 2001; Vasta et al., 2006). In PDE8A mouse knockouts, there were increased intracellular concentrations of cAMP and 4-fold higher testosterone levels compared to wild-type mice. When wild-type mice were treated with a PDE8A-specific inhibitor, there was a significant increase in steroidogenic response to LH (Wang et al., 2001). Thus, targeting PDE8A could be of interest in potentiating the endogenous effects of LH stimulation. It may be particularly effective in improving age-related hypogonadism because it has been shown in a rat model with primary hypogonadism, a major defect is decreased LH responsiveness of the aged Leydig cells and thus reduced cAMP (Chen et al., 2004). These authors showed that treatment of the aged cells with cAMP increased testosterone formation significantly. In addition to increasing cAMP © 2014 American Society of Andrology and European Academy of Andrology
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production, the use of PDE8A inhibition might prolong the halflife of cAMP through inhibiting these phosphodiesterases which are conserved in humans (Wang et al., 2001), and thus may serve to increase testosterone formation in this way as well. This approach represents a modality to specifically target a part of the steroidogenic pathway downstream of the LH receptor. Translocator protein The translocator protein, previously known as the peripheral benzodiazepine receptor, is an 18 kDa outer mitochondrial membrane protein that interacts with StAR, among other proteins, and facilitates the transfer of cholesterol to the inner mitochondrial membrane in response to ligand induction (Chung et al., 2013). TSPO disruption, through its effect on cholesterol transport into the mitochondria, thus impairs steroidogenesis (Papadopoulos et al., 1997). StAR and TSPO expressions are reduced in aged Leydig cells, contributing to decreased testosterone production (Culty et al., 2002). Recent work by Chung et al. demonstrated that in aged Brown Norway rats, increased testosterone production could be elicited in vitro by treating Leydig cells isolated from young and aged rats with TSPO ligands and also in vivo by the administration of TSPO ligand (Chung et al., 2013). The significance of this approach is that it represents a possible means by which to treat hypogonadism that does not directly rely on LH signalling. Thus, there is a possibility that this approach can be used to correct both primary and hypogonadotropic hypogonadism because it can increase testosterone production even in the absence of LH stimulation. Electron transport chain of mitochondria The central role of mitochondria in steroidogenesis provides another possible target. In a study conducted by Midzak et al., treatment of Leydig cells with the electron transport chain inhibitor myxothiazol (MYX) in vitro in the absence of LH stimulation resulted in a 300% increase in basal testosterone production in an LH-independent fashion (Midzak et al., 2007). This was an unexpected finding; the expected result was tonic inhibition of steroidogenesis, which occurred when the same cells were treated with LH and MYX. Thus, MYX produced a paradoxical effect on steroidogenesis, causing inhibition of LH-induced steroidogenesis but stimulation of LH-independent steroidogenesis. The mechanism of action, while not clear, appears to involve a redox-dependent stimulation of TSPO transport of cholesterol into the mitochondria. It also may function through increasing intracellular calcium concentrations, which leads to cAMP -independent testosterone synthesis (Sullivan & Cooke, 1986; Kumar et al., 1994; Tomic et al., 1995). Use of other electron transport chain inhibitors produced the same effect. The advantages of such an approach are that it uses small molecules that can access the Leydig cell, and seems to work independently of LH stimulation. The major disadvantages are that this approach would inhibit LH-dependent steroidogenesis, and has the potential for wide-ranging effects of electron transport chain inhibition in many cells. Cox-2 and thromboxane A synthase LH stimulation of the Leydig cell also results in arachidonic acid release by cell membrane lipids. As shown in Fig. 1, the metabolism of arachidonic acid in a COX-2-dependent fashion results in metabolites including prostaglandin H2 (PGH2), © 2014 American Society of Andrology and European Academy of Andrology
ANDROLOGY PGF2a, PGE2, PGI2 and thromboxane A2. These end-products inhibit the promoter region of the StAR gene, and thus have effects on the translocation of cholesterol into mitochondria and ultimately on steroid formation. Previous studies demonstrated that small-molecule inhibition of COX-2 and thromboxane A synthase resulted in significant increases in testosterone production by Leydig cells isolated from aged rats (Wang et al., 2008). Furthermore, when the rats were administered COX2 inhibitors in their diet, they also had increased serum testosterone levels (Wang et al., 2005, 2008). Pharmacologically, this approach is very promising because it demonstrates that orally dosed COX2 inhibitors can successfully enter the testes and exert their effects on steroidogenesis. To extrapolate further, currently available FDA-approved COX2 inhibitors such as Celebrex may warrant investigation as a treatment for hypogonadism in humans. Thromboxane A synthase (TBXAS) is an enzyme downstream of COX2 that converts prostaglandin H2 to thromboxane A2. Thromboxane A2 inhibits CREB Binding Protein from interacting with the StAR promoter region thus contributing to the tonic inhibition of StAR activation (Wang et al., 2005). Studies conducted in MA-10 mouse Leydig cells demonstrated increases in steroid production with siRNA inactivation of thromboxane A synthase. This was additionally accompanied by increases in StAR mRNA and protein expression levels. The experiment was then repeated with TBXAS small-molecule inhibitors, which resulted in the same effect (Wang et al., 2008). These results indicate that the arachidonic acid metabolic pathway has two potential targets that are upregulated with ageing, and whose inhibition in cell culture and animal models resulted in increased steroidogenesis. The clear advantages of these targets are small-molecule approaches, a relatively well-understood mechanistic pathway, and an understanding of the toxicity profile of disrupting prostaglandin production. Osteocalcin Osteocalcin, also known as bone gamma-carboxyglutamic acid-containing protein, is a natural protein product of osteoblast metabolism and is exclusively produced by osteoblasts. It undergoes vitamin K-dependent carboxylation in the synthesis of its final product. It is secreted in both the serum and urine (Lian & Gundberg, 1988; Oury et al., 2011), and is highly correlated with bone turnover (Charles et al., 1985). Normal concentrations in the adult serum range from 2 to 12 ng/mL, with a mean of 7.0 ng/mL (Gundberg et al., 1983). These concentrations remain relatively constant from age 30–60 years (Gundberg et al., 1983). In vivo, pharmacological administration of calcitriol to rats caused a 4- to 8-fold increase in serum osteocalcin (Price & Baukol, 1981). As demonstrated in Fig. 1, osteocalcin binds to a non-LH receptor, receptor Gprc6a, which bypasses the LH receptor and may directly interact with CREB to upregulate mRNA expression of StAR protein. Oury et al. (2011) described a novel interaction between bone metabolism and testosterone production through osteocalcin, which binds to a cell-membrane bound G-protein-coupled receptor 6A (Gprc6a) promoting testosterone synthesis by Leydig cells (Oury et al., 2011). In mice with osteocalcin knockout and osteocalcin gain of function, a link was shown between osteocalcin stimulation, testosterone production and male fertility. Thus, mice with gain of function mutations had larger testes, increased fertility and increased testosterone production and those with osteocalcin deficiencies Andrology, 1–7
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demonstrated the opposite (Oury et al., 2011). Although this study was conducted in mice, there is a human analogue to osteocalcin and its receptor, Gprc6a, and deficiency of this receptor was found in a recent study to be associated with infertility (Oury et al., 2013). Several recent human studies have sought to investigate the relationship between osteocalcin and testosterone production. Schwetz et al. correlated serum testosterone levels and carboxylated and uncarboxylated osteocalcin levels with sperm count in an infertile male population and found a statistically significant but weak correlation (Schwetz et al., 2013). Another study looked at bisphosphonate administration which lowers serum osteocalcin levels by ~40%, and found that it did not cause significant reductions in serum testosterone levels (Bolland et al., 2013). Other studies from Europe and Asia have shown promising, positive correlations at population levels with osteocalcin and testosterone (Kyvernitakis et al., 2013; Liao et al., 2013). Osteocalcin, a vitamin K-dependent protein, may be affected by medications such as warfarin, hydrochlorothiazide, bisphosphonates and calcitriol (Lian & Gundberg, 1988; Bolland et al., 2013). Based on these previous studies, it is possible that osteocalcin manipulation could provide a new opportunity for fertility and ED treatment, and perhaps for the treatment for hypogonadal sexual dysfunction in a way that does not adversely affect spermatogenesis (Smith & Saunders, 2011). The advantage of using the osteocalcin-testosterone regulatory loop as an approach to correct hypogonadism is that it may be done indirectly through improving bone health. However, a possible disadvantage is that targeting this loop may have unexpected consequences in other systems.
CONCLUSIONS Our understanding of the regulatory mechanisms of testosterone within the Leydig cell has greatly improved over the past two decades. Currently, pharmacologic treatments for hypogonadism are limited to those that act through the LH receptor, such as hCG, clomiphene, or aromatase inhibitors, or by bypassing endogenous production entirely through the exogenous administration of testosterone. Herein, we identified five future targets that might promote increased endogenous testosterone production through the Leydig cell instead of relying on exogenous testosterone administration. The common feature of each is that they do not rely upon the LH receptor. The potential approaches use small molecules, dietary supplements, or even FDA-approved medications. Although the data for these pharmacologic agents come primarily from animal studies, further study in human trials could prove promising and deserve further study.
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ANDROLOGY Bolland MJ, Grey A, Horne AM & Reid IR. (2013) Testosterone levels following decreases in serum osteocalcin. Calcif Tissue Int 93, 133–136. Bonavera JJ, Swerdloff RS, Leung A, Lue YH, Baravarian S, Superlano L, Sinha-Hikim AP & Wang C. (1997) In the male brown-Norway (BN) male rat, reproductive aging is associated with decreased LH-pulse amplitude and area. J Androl 18, 359–365. Bonavera JJ, Swerdloff RS, Sinha Hakim AP, Lue YH & Wang C. (1998) Aging results in attenuated gonadotropin releasing hormone-luteinizing hormone axis responsiveness to glutamate receptor agonist N-methyl-D-aspartate. J Neuroendocrinol 10, 93–99. Bremner WJ, Millar MR, Sharpe RM & Saunders PT. (1994) Immunohistochemical localization of androgen receptors in the rat testis: evidence for stage-dependent expression and regulation by androgens. Endocrinology 135, 1227–1234. Charles P, Poser JW, Mosekilde L & Jensen FT. (1985) Estimation of bone turnover evaluated by 47Ca-kinetics. Efficiency of serum bone gamma-carboxyglutamic acid-containing protein, serum alkaline phosphatase, and urinary hydroxyproline excretion. J Clin Invest 76, 2254–2258. Chen H, Hardy MP & Zirkin BR. (2002) Age-related decreases in Leydig cell testosterone production are not restored by exposure to LH in vitro. Endocrinology 143, 1637–1642. Chen H, Liu J, Luo L & Zirkin BR. (2004) Dibutyryl cyclic adenosine monophosphate restores the ability of aged Leydig cells to produce testosterone at the high levels characteristic of young cells. Endocrinology 145, 4441–4446. Chung JY, Chen H, Midzak A, Burnett AL, Papadopoulos V & Zirkin BR. (2013) Drug ligand-induced activation of translocator protein (TSPO) stimulates steroid production by aged brown Norway rat Leydig cells. Endocrinology 154, 2156–2165. Cole LA. (2010) Biological functions of hCG and hCG-related molecules. Reprod Biol Endocrinol 8, 102. Conn PM & Crowley WF Jr. (1991) Gonadotropin-releasing hormone and its analogues. N Engl J Med 324, 93–103. Corona G, Vignozzi L, Sforza A & Maggi M. (2013) Risks and benefits of late onset hypogonadism treatment: an expert opinion. World J Mens Health 31, 103–125. Culty M, Luo L, Yao ZX, Chen H, Papadopoulos V & Zirkin BR. (2002) Cholesterol transport, peripheral benzodiazepine receptor, and steroidogenesis in aging Leydig cells. J Androl 23, 439–447. Donohue JM, Cevasco M & Rosenthal MB. (2007) A decade of direct-to-consumer advertising of prescription drugs. N Engl J Med 357, 673–681. Duke VM, Winyard PJ, Thorogood P, Soothill P, Bouloux PM & Woolf AS. (1995) KAL, a gene mutated in Kallmann’s syndrome, is expressed in the first trimester of human development. Mol Cell Endocrinol 110, 73–79. Gruenewald DA & Matsumoto AM. (2003) Testosterone supplementation therapy for older men: potential benefits and risks. J Am Geriatr Soc 51, 101–115; discussion 115. Grzywacz FW, Chen H, Allegretti J & Zirkin BR. (1998) Does age-associated reduced Leydig cell testosterone production in Brown Norway rats result from under-stimulation by luteinizing hormone? J Androl 19, 625–630. Gundberg CM, Lian JB & Gallop PM. (1983) Measurements of gamma-carboxyglutamate and circulating osteocalcin in normal children and adults. Clin Chim Acta 128, 1–8. Hamada AJ, Montgomery B & Agarwal A. (2012) Male infertility: a critical review of pharmacologic management. Expert Opin Pharmacother 13, 2511–2531. Harman SM, Metter EJ, Tobin JD, Pearson J & Blackman MR. (2001) Longitudinal effects of aging on serum total and free testosterone levels in healthy men. Baltimore Longitudinal Study of Aging. J Clin Endocrinol Metab 86, 724–731.
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TARGETS FOR ENDOGENOUS TESTOSTERONE PRODUCTION Hotaling JM & Patel Z. (2014) Male endocrine dysfunction. Urol Clin North Am 41, 39–53. Hussein A, Ozgok Y, Ross L & Niederberger C. (2005) Clomiphene administration for cases of nonobstructive azoospermia: a multicenter study. J Androl 26,787–791; discussion 792-3. Jarow JP & Zirkin BR. (2005) The androgen microenvironment of the human testis and hormonal control of spermatogenesis. Ann N Y Acad Sci 1061, 208–220. Jarow JP, Chen H, Rosner TW, Trentacoste S & Zirkin BR. (2001) Assessment of the androgen environment within the human testis: minimally invasive method to obtain intratesticular fluid. J Androl 22, 640–645. Kamischke A & Nieschlag E. (1999) Analysis of medical treatment of male infertility. Hum Reprod 14(Suppl 1), 1–23. Katz DJ, Nabulsi O, Tal R & Mulhall JP. (2012) Outcomes of clomiphene citrate treatment in young hypogonadal men. BJU Int 110, 573–578. Kumar S, Davies M, Zakaria Y, Mawer EB, Gordon C, Olukoga AO & Boulton AJ. (1994) Improvement in glucose tolerance and beta-cell function in a patient with vitamin D deficiency during treatment with vitamin D. Postgrad Med J 70, 440–443. Kyvernitakis I, Saeger U, Ziller V, Bauer T, Seker-Pektas B & Hadji P. (2013) The effect of age, sex hormones, and bone turnover markers on calcaneal quantitative ultrasonometry in healthy German men. J Clin Densitom 16, 320–328. Layman LC, Cohen DP, Xie J & Smith GD. (2002) Clinical phenotype and infertility treatment in a male with hypogonadotropic hypogonadism due to mutations Ala129Asp/Arg262Gln of the gonadotropin-releasing hormone receptor. Fertil Steril 78, 1317–1320. Leder BZ, Rohrer JL, Rubin SD, Gallo J & Longcope C. (2004) Effects of aromatase inhibition in elderly men with low or borderline-low serum testosterone levels. J Clin Endocrinol Metab 89, 1174–1180. Lian JB & Gundberg CM. (1988) Osteocalcin. Biochemical considerations and clinical applications. Clin Orthop Relat Res, 267–291. Liao M, Guo X, Yu X, Pang G, Zhang S, Li J, Tan A, Gao Y, Yang X, Zhang H, Qin X, Mo L, Lu Z, Wu C & Mo Z. (2013) Role of metabolic factors in the association between osteocalcin and testosterone in chinese men. J Clin Endocrinol Metab 98, 3463–3469. Liu PY, Iranmanesh A, Nehra AX, Keenan DM & Veldhuis JD. (2005a) Mechanisms of hypoandrogenemia in healthy aging men. Endocrinol Metab Clin North Am 34, 935–955, ix. Liu PY, Takahashi PY, Roebuck PD, Iranmanesh A & Veldhuis JD. (2005b) Aging in healthy men impairs recombinant human luteinizing hormone (LH)-stimulated testosterone secretion monitored under a two-day intravenous pulsatile LH clamp. J Clin Endocrinol Metab 90, 5544–5550. Manna PR, Dyson MT & Stocco DM. (2009) Regulation of the steroidogenic acute regulatory protein gene expression: present and future perspectives. Mol Hum Reprod 15, 321–333. Midzak AS, Liu J, Zirkin BR & Chen H. (2007) Effect of myxothiazol on Leydig cell steroidogenesis: inhibition of luteinizing hormone-mediated testosterone synthesis but stimulation of basal steroidogenesis. Endocrinology 148, 2583–2590. Midzak AS, Chen H, Papadopoulos V & Zirkin BR. (2009) Leydig cell aging and the mechanisms of reduced testosterone synthesis. Mol Cell Endocrinol 299, 23–31. Oury F, Sumara G, Sumara O, Ferron M, Chang H, Smith CE, Hermo L, Suarez S, Roth BL, Ducy P & Karsenty G. (2011) Endocrine regulation of male fertility by the skeleton. Cell 144, 796–809. Oury F, Ferron M, Huizhen W, Confavreux C, Xu L, Lacombe J, Srinivas P, Chamouni A, Lugani F, Lejeune H, Kumar TR, Plotton I & Karsenty G. (2013) Osteocalcin regulates murine and human fertility through a pancreas-bone-testis axis. J Clin Invest 123, 2421– 2433. Page ST. (2011) Physiologic role and regulation of intratesticular sex steroids. Curr Opin Endocrinol Diabetes Obes 18, 217–223.
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ANDROLOGY Papadopoulos V & Miller WL. (2012) Role of mitochondria in steroidogenesis. Best Pract Res Clin Endocrinol Metab 26, 771–790. Papadopoulos V, Amri H, Li H, Boujrad N, Vidic B & Garnier M. (1997) Targeted disruption of the peripheral-type benzodiazepine receptor gene inhibits steroidogenesis in the R2C Leydig tumor cell line. J Biol Chem 272, 32129–32135. Pierce JG & Parsons TF. (1981) Glycoprotein hormones: structure and function. Annu Rev Biochem 50, 465–495. Price PA & Baukol SA. (1981) 1,25-dihydroxyvitamin D3 increases serum levels of the vitamin K-dependent bone protein. Biochem Biophys Res Commun 99, 928–935. Ramasamy R, Stahl PJ & Schlegel PN. (2012) Medical therapy for spermatogenic failure. Asian J Androl 14, 57–60. Reifsnyder JE, Ramasamy R, Husseini J & Schlegel PN. (2012) Role of optimizing testosterone before microdissection testicular sperm extraction in men with nonobstructive azoospermia. J Urol 188, 532–536. Schwetz V, Gumpold R, Graupp M, Hacker N, Schweighofer N, Trummer O, Pieber TR, Ballon M, Lerchbaum E & Obermayer-Pietsch B. (2013) Osteocalcin is not a strong determinant of serum testosterone and sperm count in men from infertile couples. Androl 1, 590–594. Shinjo E, Shiraishi K & Matsuyama H. (2013) The effect of human chorionic gonadotropin-based hormonal therapy on intratesticular testosterone levels and spermatogonial DNA synthesis in men with non-obstructive azoospermia. Androl 1, 929–935. Sih R, Morley JE, Kaiser FE, Perry HM III, Patrick P & Ross C. (1997) Testosterone replacement in older hypogonadal men: a 12-month randomized controlled trial. J Clin Endocrinol Metab 82, 1661–1667. Smith LB & Saunders PT. (2011) The skeleton: the new controller of male fertility? Cell 144, 642–643. Sullivan MH & Cooke BA. (1986) The role of Ca2+ in steroidogenesis in Leydig cells. Stimulation of intracellular free Ca2+ by lutropin (LH), luliberin (LHRH) agonist and cyclic AMP. Biochem J 236, 45–51. Tomic M, Dufau ML, Catt KJ & Stojilkovic SS. (1995) Calcium signaling in single rat Leydig cells. Endocrinology 136, 3422–3429. Trussell JC. (2013) Male reproductive endocrinology: when to replace gonadotropins. Semin Reprod Med 31, 237–244. Tsai LC & Beavo JA. (2011) The roles of cyclic nucleotide phosphodiesterases (PDEs) in steroidogenesis. Curr Opin Pharmacol 11, 670–675. Vasta V, Shimizu-Albergine M & Beavo JA. (2006) Modulation of Leydig cell function by cyclic nucleotide phosphodiesterase 8A. Proc Natl Acad Sci U S A 103, 19925–19930. Wang P, Wu P, Egan RW & Billah MM. (2001) Human phosphodiesterase 8A splice variants: cloning, gene organization, and tissue distribution. Gene 280, 183–194. Wang X, Shen CL, Dyson MT, Eimerl S, Orly J, Hutson JC & Stocco DM. (2005) Cyclooxygenase-2 regulation of the age-related decline in testosterone biosynthesis. Endocrinology 146, 4202–4208. Wang X, Yin X, Schiffer RB, King SR, Stocco DM & Grammas P. (2008) Inhibition of thromboxane a synthase activity enhances steroidogenesis and steroidogenic acute regulatory gene expression in MA-10 mouse Leydig cells. Endocrinology 149, 851–857. Zirkin BR & Chen H. (2000) Regulation of Leydig cell steroidogenic function during aging. Biol Reprod 63, 977–981. Zirkin BR, Santulli R, Awoniyi CA & Ewing LL. (1989) Maintenance of advanced spermatogenic cells in the adult rat testis: quantitative relationship to testosterone concentration within the testis. Endocrinology 124, 3043–3049. Zwart AD, Urban RJ, Odell WD & Veldhuis JD. (1996) Contrasts in the gonadotropin-releasing hormone dose-response relationships for luteinizing hormone, follicle-stimulating hormone and alpha-subunit release in young versus older men: appraisal with high-specificity immunoradiometric assay and deconvolution analysis. Eur J Endocrinol 135, 399–406.
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ISSN: 2047-2919
REVIEW ARTICLE
Correspondence: Brian Le, The James Buchanan Brady Urological Institute, Johns Hopkins Hospital, 600 North Wolfe Street, Marburg 145 Baltimore, MD 21287-2101, USA. E-mail: [email protected]
Keywords: ageing, androgens, Leydig cells, LH, LH receptor, testosterone
New targets for increasing endogenous testosterone production: clinical implications and review of the literature 1
B. Le, 2H. Chen, 2B. Zirkin and 1A. Burnett
1
Received: 3-Dec-2013 Revised: 3-Mar-2014 Accepted: 6-Apr-2014
The James Buchanan Brady Urological Institute, Johns Hopkins Hospital, Baltimore, MD, USA, and Department of Biochemistry and Molecular Biology, The Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, USA 2
doi: 10.1111/j.2047-2927.2014.00225.x
SUMMARY Over the past several decades, our understanding of the regulatory mechanisms of testosterone production has increased significantly. Concurrently, the medical treatment of hypogonadism, particularly in the ageing male has increased. This review article consolidates some of our insights into the regulatory mechanisms of endogenous testosterone production and examines promising new targets that may allow endogenous production of testosterone to be re-established in males with primary hypogonadism. We examined the published scientific literature regarding regulatory mechanisms of testosterone biosynthesis with a focus on Leydig cell physiology and small-molecule regulation that resulted in increased testosterone production. We identified several pathways that have been manipulated pharmacologically to increase Leydig cell testosterone production, including phosphodiesterases, the cholesterol translocator protein, the electron transport chain of mitochondria, cyclooxygenases and osteocalcin. Manipulation of these pathways with small molecules has helped further our understanding of the regulatory pathways of testosterone biosynthesis. Herein, we identified five future targets that might promote increased endogenous testosterone production through the Leydig cell instead of relying on exogenous testosterone administration.
INTRODUCTION In recent years, public awareness of the signs and symptoms of low serum testosterone or hypogonadism has increased. Although they are often vague, these include common symptoms of fatigue, worsening cognition, decreased libido, decreased muscle mass, depressed mood and sexual dysfunction (Bassil, 2011). Low serum testosterone, the end result of decreased endogenous production and bioavailability of testosterone, is believed to affect ~5 million American men (Araujo et al., 2007). Clinically, physicians see two main presentations for hypogonadism: (i) the younger male presenting with infertility or subfertility, and (ii) the older male with signs and symptoms of low testosterone who is seeking evaluation. In the first scenario, a history, physical examination and a panel of tests (including semen analysis and serum levels of FSH, LH, testosterone, estradiol and prolactin) are performed to determine whether hypogonadism is associated with, and perhaps the cause of the male infertility, as opposed to other causes such as obstruction or genetic abnormalities. If low intratesticular testosterone (ITT) is a suspected aetiology, which would be © 2014 American Society of Andrology and European Academy of Andrology
suggested by low serum testosterone levels, LH levels are examined to help differentiate between hypogonadotropic hypogonadism (low LH, low T) vs. primary hypogonadism (high LH, low T). Hypogonadotropic hypogonadism is the rarer of the two entities and is associated with Kallman syndrome, idiopathic hypogonadotropic hypogonadism and congenital adrenal hyperplasia (Duke et al., 1995; Layman et al., 2002; Trussell, 2013). In the case of hypogonadotropic hypogonadism, treatment is directed towards stimulation of the Leydig cell through the LH receptor by pharmacologic means [recombinant LH, hCG, pulsatile gonadotropin releasing hormone (GnRH), or clomiphene], the objective is to correct putative low intratesticular testosterone levels and thus restore spermatogenesis (Hamada et al., 2012). Primary hypogonadism, the more common entity, can be more challenging to manage, but some may still respond to LH receptor stimulation (Hussein et al., 2005; Trussell, 2013; Hotaling & Patel, 2014). Hypogonadism has been shown to be associated with nonobstructive azoospermia in as many as 47% of patients undergoing testicular sperm extraction, and therefore means by which Andrology, 1–7
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to modulate testosterone levels have been areas of extreme interest (Ramasamy et al., 2012). Unfortunately, treatment with exogenous LH/hCG/clomiphene in individuals with primary hypogonadism is often ineffective in stimulating Leydig cell testosterone production to raise serum and intratesticular testosterone levels (Bremner et al., 1994; Zwart et al., 1996; Hussein et al., 2005; Reifsnyder et al., 2012; Trussell, 2013). Moreover, the exogenous administration of testosterone itself, although it increases serum testosterone levels, is unlikely to increase intratesticular testosterone levels. Rather, such treatment is likely to reduce these levels by negative feedback inhibition of pituitary LH secretion (Kamischke & Nieschlag, 1999). This presents a conundrum for the clinician who seeks to increase intratesticular testosterone, but has few options that do not act through the LH receptor. In the second scenario, that of the older hypogonadal male, he also suffers from the problem of decreased Leydig cell responsiveness to LH stimulation and low endogenous testosterone production, and is thus a form of primary hypogonadism. Primary hypogonadism is the predominant type found in this population (Sih et al., 1997; Harman et al., 2001). In men, serum testosterone levels typically begin to decline in the fifth decade of life and are usually accompanied by increasing serum levels of FSH and stable or increased levels of LH (Zirkin & Chen, 2000; Midzak et al., 2009). However, in this situation, the men are usually less concerned about fertility and so they routinely turn to exogenous administration of testosterone. This is by far the larger target market for pharmaceutical companies, and over the past several years, there has been increased marketing by such companies. Indeed, advertising expenditures on exogenous testosterone supplementation have increased 1000% from 2010 to 2012 (Donohue et al., 2007). Correction of testosterone levels of less than 300 ng/dL in symptomatic men often leads to symptomatic improvement and improved quality of life (Sih et al., 1997; Gruenewald & Matsumoto, 2003). Furthermore, developments in delivery systems including gels, roll-on applications and subcutaneous pellets, all of which bypass first pass metabolism in the liver, have made testosterone replacement increasingly accessible and palatable to men. However, exogenous testosterone replacement is not without its risks and drawbacks, including increased risk of cardiovascular events, prostate growth, worsening sleep apnoea, testicular atrophy, infertility and exacerbation of certain types of cancers (Corona et al., 2013). Additionally, the use of exogenous testosterone ultimately leads to LH down-regulation and shutdown of endogenous production of testosterone and thus reductions in intratesticular testosterone levels that are essential for spermatogenesis (Zirkin et al., 1989). The long-term consequences of exogenous testosterone supplementation on the intratesticular environment are not entirely clear. This review article examines promising new targets that may allow endogenous production of testosterone to be reestablished in males with primary hypogonadism, and thus increase intratesticular testosterone levels. The objectives of such approaches should be to: (i) maintain an intratesticular testosterone environment that allows/promotes normal spermatogenesis, (ii) avoid side effects of exogenous testosterone supplementation and (iii) maintain the negative feedback mechanism of LH that prevents testosterone levels from becoming supratherapeutic. We review new potential treatments that have 2
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ANDROLOGY these advantages, and discuss the clinical promise of such findings.
INTRATESTICULAR TESTOSTERONE LEVELS The focus on intratesticular testosterone levels as opposed to serum testosterone levels is an important distinction. It is well established that the total testosterone concentration within the testes is far higher than that in the serum, with intratesticular testosterone levels 8- to 30-fold higher in rodents and 20- to 100-fold higher in humans (Jarow & Zirkin, 2005). However, the relationship between serum testosterone levels and intratesticular testosterone levels is non-linear and has considerable variability. For example, Jarow et al., using percutaneous aspiration of the testes in fertile men in a clinical setting, determined that the mean total testosterone concentration within the testes was 600 ng/mL, compared to 5 ng/mL in serum, but with considerable variability between individuals (Jarow et al., 2001). The aetiology of this concentration differential is likely because of local testosterone production by Leydig cells and the countercurrent mechanism in the pampiniform plexus (Jarow & Zirkin, 2005). When clinically low serum testosterone levels are detected, it may be the end result of the disruption of steroidogenic production by Leydig cells, although other explanations are possible. Testosterone mediates its effects on spermatogenesis by binding to the androgen receptor found on Sertoli cells, and also binds to receptors in Leydig and peritubular cells (Bremner et al., 1994; Page, 2011). Under normal conditions, high intratesticular concentrations of testosterone are a requirement for the maintenance of normal spermatogenesis and thus the prevention of testicular atrophy. For example, in the rat reductions of intratesticular testosterone levels to 20% of normal values do not seem to affect spermatogenesis, but reductions below this level result in dramatic decreases in sperm production. However, 20% of normal intratesticular testosterone represents a far higher level (4- to 5-fold) than normal serum levels (Zirkin et al., 1989). Data such as these suggest an association between hypogonadism and male infertility in young men that may be treated with correction of intratesticular testosterone levels. However, the exact relationship between endocrine dysfunction, intratesticular testosterone levels and infertility are poorly understood. This is further complicated as male infertility is a heterogenous entity with presentations from azoospermia to subfertility with normal semen parameters. Indeed only 3% of male infertility cases have an endocrine aetiology, as measured by serum hormone values, and identified as a treatable cause of infertility, despite 30–70% of infertile men having some degree of endocrine dysfunction (Hotaling & Patel, 2014). There are very limited data on ITT in the setting of male infertility from humans. In many cases, ITT levels may be normal or increased in patients with non-obstructive azoospermia at the time of testicular sperm extraction which is believed to be owing to a primary genetic defect with endocrine compensation (Shinjo et al., 2013).
PATHOPHYSIOLOGY OF DECREASED TESTOSTERONE PRODUCTION IN THE AGEING MALE Cross-sectional and longitudinal ageing studies have demonstrated that ageing is associated with a progressive decline in serum testosterone levels (Harman et al., 2001). The prevalence of clinically symptomatic low testosterone is estimated to be © 2014 American Society of Andrology and European Academy of Andrology
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20% of men at age 60, and 50% at age 80 (Gruenewald & Matsumoto, 2003). Despite these decreases in serum testosterone, LH levels are usually unchanged or increased, while FSH levels are usually increased compared to younger males, suggesting a primary testicular deficiency (Zwart et al., 1996). Although agerelated changes in GnRH gene expression and the amplitude of LH pulses have been shown as well, this is believed to be less important than decreased Leydig cell responsiveness of LH, which has been demonstrated in rats (Bonavera et al., 1997, 1998) as well as in humans (Liu et al., 2005a,b). In a study conducted by Liu et al., they administered recombinant human LH in a pulsatile fashion to healthy young and old men and examined the steroidogenic response. They noted a blunted testosterone peak, amplitude and response to stimulation compared to younger men (Liu et al., 2005a,b). Studies examining the differences between young and aged Leydig cells that have come primarily from the study of rat models of ageing have elucidated several key differences including: (i) decreased ability to synthesize and mobilize cholesterol for steroidogenesis by aged Leydig cells (ii) decreased number of LH receptors, (iii) decreased activity and expression of cholesterol side-chain cleavage enzyme (CYP11A1) and (iv) decreased activity of steroidogenic enzymes in the smooth endoplasmic reticulum involved in converting pregnenolone to testosterone (Zirkin & Chen, 2000).
CURRENT APPROACHES Current approaches to the treatment of male hypogonadism begin with a careful clinical history and physical examination to identify agents or aetiologies that may be adversely affecting steroidogenesis and spermatogenesis, such as anabolic steroid use, medications, presence of varicocoeles and trauma. When possible, these conditions are treated. The aetiology may be identified as a distal (pituitary/hypothalamus/exogenous) or a local (testicular) deficiency, termed secondary and primary hypogonadism, respectively. In these cases, clinicians turn to pharmacologic means to stimulate testosterone production, or to provide testosterone via exogenous administration so as to counter the systemic effects of hypogonadism. Pharmacologic attempts at enhancing endogenous production of testosterone have been at best modestly effective, and only in select conditions, notably clearly identifiable deficiencies in central stimulation of otherwise normal testes (Bobjer et al., 2012). One current approach is to manipulate the hypothalamic–pituitary–gonadal (HPG) axis. Under normal conditions, the hypothalamus releases gonadotropin releasing hormone (GnRH) which stimulates the anterior pituitary to release FSH and LH in a pulsatile fashion (Conn & Crowley, 1991). LH passes through the circulation and binds to the LH receptor located on Leydig cells to stimulate production of testosterone; FSH binds to Sertoli cells which support spermatogenesis. For aetiologies of male infertility, such as in Kallman’s syndrome or hypogonadotropic hypogonadism, deficiencies in LH production can be overcome by pharmacologic activation of LH or the LH receptor (Hamada et al., 2012). There are two main ways this is accomplished: direct stimulation of testosterone production by administering LH or hCG, and indirect effects on testosterone production with clomiphene or aromatase inhibitors (Page, 2011). With direct stimulation, recombinant LH is injected into the bloodstream and binds directly to the Leydig cell LH receptor to stimulate the Leydig cells to produce testosterone. With © 2014 American Society of Andrology and European Academy of Andrology
ANDROLOGY exogenous administration of hCG, the common alpha-subunit is homologous to LH, while the beta-subunit has considerable similarity but distinguishes hCG from LH (Pierce & Parsons, 1981). When administered by injection, hCG also directly stimulates the LH receptor, and promotes testosterone production. The main difference between LH and hCG is their half-lives, with LH having a circulating half-life of 25–30 min compared to hCG with a half-life of 37 h. This difference is the reason that hCG is typically used clinically for hypogonadism (Katz et al., 2012) rather than recombinant LH (Cole, 2010). With indirect stimulation, the pharmacologic approach is to stimulate endogenous LH production by inhibiting negative feedback on the HPG axis. Clomiphene has been used for decades in women to regulate or induce ovulation, and has FDA approval for that purpose (Katz et al., 2012). In men, clomiphene is used ‘off-label’ to treat hypogonadism and male infertility. It acts by preventing the binding of oestrogen, which is peripherally converted from testosterone, to receptors in the hypothalamus. This inhibition of negative feedback leads to increased GnRH stimulation of the pituitary and increased LH secretion, resulting in downstream stimulation of Leydig cells (Katz et al., 2012). An alternative approach that is used is to administer selective aromatase inhibitors which block the peripheral conversion of testosterone to estradiol through the process of aromatization (Leder et al., 2004). This again inhibits the negative feedback mechanism of the HPG axis, thus promoting downstream endogenous testosterone production. Recombinant LH, hCG, clomiphene and aromatase inhibitors all act directly or indirectly through stimulation of the LH receptor located on Leydig cells. However, in patients with normal or high LH levels and persistent hypogonadism, increasing stimulation generally does not result in increased testosterone production. In fact, hypogonadism in the vast majority of patients is not because of central deficiencies (Ramasamy et al., 2012), but rather is due to decreased responsiveness of the Leydig cell to LH (Grzywacz et al., 1998; Midzak et al., 2009). Thus, methods that aim to enhance testosterone production through LH stimulation are inherently limited, and in particular have not been very effective clinically in the ageing population (Chen et al., 2002). Clearly, there is a need to examine alternative approaches to increasing intratesticular testosterone levels and endogenous production.
FUTURE TARGETS AND APPROACHES Discussions of the possibility of identifying LH-independent mechanisms by which to increase testosterone inevitably must start with an understanding of how testosterone production by Leydig cells is regulated normally, and what is right and wrong with these cells when they no longer respond well to LH. The mechanism of LH receptor stimulation of Leydig cell testosterone biosynthesis has been an area of intense study. Figure 1 highlights pathways involved in cell signalling related to testosterone biosynthesis that have small-molecule targets. There are additional regulatory mechanisms that have been simplified for clarity. Stimulation of Leydig cell testosterone biosynthesis involves the following: LH receptor stimulation leading to cAMP formation; activation of protein kinase A; phosphorylation of transcription factors GATA-4 and CREB; regulation of interaction of StAR and translocator protein (TSPO), leading to translocation Andrology, 1–7
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Figure 1 Signalling pathways involved in Leydig Cell steroidogenesis and potential targets for enhancing testosterone production (in bold). Legend: ATP, Adenosine Triphosphate; cAMP, Cyclic Adenosine Monophosphate; CBP, CREB Binding Protein; CREB, cAMP Response Element-Binding Protein; COX-2, Cyclooxygenase 2; COX2i, Cyclooxygenase 2 inhibitor; GATA-4, Transcription Factor GATA4; GPRC6A, G-protein-coupled receptor encoded by GPRC6A; hCG, Human Chorionic Gonadotropin; LH, Luteinizing Hormone; LH-R, LH Receptor; PKA, Protein Kinase A; PKAc, Protein Kinase A Catalytic subunit; SF-1, Steroidogenic Factor 1; stAR, Steroidogenic Acute Regulatory Protein; TBXAS inhibitor, Thromboxane A Synthase inhibitor; TSPO, Translocator Protein.
of cholesterol into the mitochondria (Manna et al., 2009; Papadopoulos & Miller, 2012); conversion of cholesterol to pregnenolone in the mitochondria; and translocation of pregnenolone to the smooth endoplasmic reticulum to be converted into testosterone and secreted by the Leydig cell (Fig. 1). The rate-limiting step in this pathway is the translocation of cholesterol from intracellular stores to the inner mitochondrial membrane. Two proteins that are critical to this translocation are StAR and the cholesterol TSPO (Zirkin & Chen, 2000; Culty et al., 2002). The reasons for the decline in testosterone biosynthesis under conditions of primary hypogonadism are numerous. Several key observations have been noted: decreased responsiveness of the LH receptor; decreases in the StAR protein; decreases in TSPO; increases in COX2 expression; reduced conversion of cholesterol to pregnenolone in the mitochondria; and reduced conversion of pregnenolone ultimately to testosterone in the smooth endoplasmic reticulum. Together these observations point to downstream deficiencies in signalling that converge on cholesterol translocation into the mitochondria, the rate-limiting step in testosterone formation and thus on the StAR protein and on TSPO, both integrally involved in cholesterol transfer to the inner mitochondrial membrane (Tsai & Beavo, 2011; Chung et al., 2013). Based on these understandings, the following describes several potential targets for increasing endogenous testosterone production that might be of particular clinical promise given the pharmacology, the strength of the data, and toxicity profiles. Furthermore, it might be possible to use these agents in concert to produce a cocktail to rescue deficient testosterone steroidogenesis. 4
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Phosphodiesterases LH binds to LH receptors which, in turn, couple to G proteins, stimulating adenylyl cyclase (AC) to convert ATP into cAMP. This is followed by activation of protein kinase A, which initiates transcriptional events that produce the steroidogenic proteins which, in Leydig cells, form testosterone from cholesterol (Fig. 1). The production of cAMP is of critical importance in testosterone formation. Phosphodiesterases (PDEs) play an important role in regulating the balance of cyclic nucleotides. The level of cAMP is determined by the rates of synthesis by ACs and degradation by PDEs (Tsai & Beavo, 2011). There are several subtypes of PDE. The main types involved in steroidogenesis by Leydig cells are those that mediate cAMP conversion to 5’AMP, including PDE8A, PDE4 and PDE2A. The most promising of these is PDE8A, which hydrolyzes cAMP exclusively and is found in high levels in the testes, spleen, colon, small intestine, ovary, placenta and kidney (Wang et al., 2001; Vasta et al., 2006). In PDE8A mouse knockouts, there were increased intracellular concentrations of cAMP and 4-fold higher testosterone levels compared to wild-type mice. When wild-type mice were treated with a PDE8A-specific inhibitor, there was a significant increase in steroidogenic response to LH (Wang et al., 2001). Thus, targeting PDE8A could be of interest in potentiating the endogenous effects of LH stimulation. It may be particularly effective in improving age-related hypogonadism because it has been shown in a rat model with primary hypogonadism, a major defect is decreased LH responsiveness of the aged Leydig cells and thus reduced cAMP (Chen et al., 2004). These authors showed that treatment of the aged cells with cAMP increased testosterone formation significantly. In addition to increasing cAMP © 2014 American Society of Andrology and European Academy of Andrology
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production, the use of PDE8A inhibition might prolong the halflife of cAMP through inhibiting these phosphodiesterases which are conserved in humans (Wang et al., 2001), and thus may serve to increase testosterone formation in this way as well. This approach represents a modality to specifically target a part of the steroidogenic pathway downstream of the LH receptor. Translocator protein The translocator protein, previously known as the peripheral benzodiazepine receptor, is an 18 kDa outer mitochondrial membrane protein that interacts with StAR, among other proteins, and facilitates the transfer of cholesterol to the inner mitochondrial membrane in response to ligand induction (Chung et al., 2013). TSPO disruption, through its effect on cholesterol transport into the mitochondria, thus impairs steroidogenesis (Papadopoulos et al., 1997). StAR and TSPO expressions are reduced in aged Leydig cells, contributing to decreased testosterone production (Culty et al., 2002). Recent work by Chung et al. demonstrated that in aged Brown Norway rats, increased testosterone production could be elicited in vitro by treating Leydig cells isolated from young and aged rats with TSPO ligands and also in vivo by the administration of TSPO ligand (Chung et al., 2013). The significance of this approach is that it represents a possible means by which to treat hypogonadism that does not directly rely on LH signalling. Thus, there is a possibility that this approach can be used to correct both primary and hypogonadotropic hypogonadism because it can increase testosterone production even in the absence of LH stimulation. Electron transport chain of mitochondria The central role of mitochondria in steroidogenesis provides another possible target. In a study conducted by Midzak et al., treatment of Leydig cells with the electron transport chain inhibitor myxothiazol (MYX) in vitro in the absence of LH stimulation resulted in a 300% increase in basal testosterone production in an LH-independent fashion (Midzak et al., 2007). This was an unexpected finding; the expected result was tonic inhibition of steroidogenesis, which occurred when the same cells were treated with LH and MYX. Thus, MYX produced a paradoxical effect on steroidogenesis, causing inhibition of LH-induced steroidogenesis but stimulation of LH-independent steroidogenesis. The mechanism of action, while not clear, appears to involve a redox-dependent stimulation of TSPO transport of cholesterol into the mitochondria. It also may function through increasing intracellular calcium concentrations, which leads to cAMP -independent testosterone synthesis (Sullivan & Cooke, 1986; Kumar et al., 1994; Tomic et al., 1995). Use of other electron transport chain inhibitors produced the same effect. The advantages of such an approach are that it uses small molecules that can access the Leydig cell, and seems to work independently of LH stimulation. The major disadvantages are that this approach would inhibit LH-dependent steroidogenesis, and has the potential for wide-ranging effects of electron transport chain inhibition in many cells. Cox-2 and thromboxane A synthase LH stimulation of the Leydig cell also results in arachidonic acid release by cell membrane lipids. As shown in Fig. 1, the metabolism of arachidonic acid in a COX-2-dependent fashion results in metabolites including prostaglandin H2 (PGH2), © 2014 American Society of Andrology and European Academy of Andrology
ANDROLOGY PGF2a, PGE2, PGI2 and thromboxane A2. These end-products inhibit the promoter region of the StAR gene, and thus have effects on the translocation of cholesterol into mitochondria and ultimately on steroid formation. Previous studies demonstrated that small-molecule inhibition of COX-2 and thromboxane A synthase resulted in significant increases in testosterone production by Leydig cells isolated from aged rats (Wang et al., 2008). Furthermore, when the rats were administered COX2 inhibitors in their diet, they also had increased serum testosterone levels (Wang et al., 2005, 2008). Pharmacologically, this approach is very promising because it demonstrates that orally dosed COX2 inhibitors can successfully enter the testes and exert their effects on steroidogenesis. To extrapolate further, currently available FDA-approved COX2 inhibitors such as Celebrex may warrant investigation as a treatment for hypogonadism in humans. Thromboxane A synthase (TBXAS) is an enzyme downstream of COX2 that converts prostaglandin H2 to thromboxane A2. Thromboxane A2 inhibits CREB Binding Protein from interacting with the StAR promoter region thus contributing to the tonic inhibition of StAR activation (Wang et al., 2005). Studies conducted in MA-10 mouse Leydig cells demonstrated increases in steroid production with siRNA inactivation of thromboxane A synthase. This was additionally accompanied by increases in StAR mRNA and protein expression levels. The experiment was then repeated with TBXAS small-molecule inhibitors, which resulted in the same effect (Wang et al., 2008). These results indicate that the arachidonic acid metabolic pathway has two potential targets that are upregulated with ageing, and whose inhibition in cell culture and animal models resulted in increased steroidogenesis. The clear advantages of these targets are small-molecule approaches, a relatively well-understood mechanistic pathway, and an understanding of the toxicity profile of disrupting prostaglandin production. Osteocalcin Osteocalcin, also known as bone gamma-carboxyglutamic acid-containing protein, is a natural protein product of osteoblast metabolism and is exclusively produced by osteoblasts. It undergoes vitamin K-dependent carboxylation in the synthesis of its final product. It is secreted in both the serum and urine (Lian & Gundberg, 1988; Oury et al., 2011), and is highly correlated with bone turnover (Charles et al., 1985). Normal concentrations in the adult serum range from 2 to 12 ng/mL, with a mean of 7.0 ng/mL (Gundberg et al., 1983). These concentrations remain relatively constant from age 30–60 years (Gundberg et al., 1983). In vivo, pharmacological administration of calcitriol to rats caused a 4- to 8-fold increase in serum osteocalcin (Price & Baukol, 1981). As demonstrated in Fig. 1, osteocalcin binds to a non-LH receptor, receptor Gprc6a, which bypasses the LH receptor and may directly interact with CREB to upregulate mRNA expression of StAR protein. Oury et al. (2011) described a novel interaction between bone metabolism and testosterone production through osteocalcin, which binds to a cell-membrane bound G-protein-coupled receptor 6A (Gprc6a) promoting testosterone synthesis by Leydig cells (Oury et al., 2011). In mice with osteocalcin knockout and osteocalcin gain of function, a link was shown between osteocalcin stimulation, testosterone production and male fertility. Thus, mice with gain of function mutations had larger testes, increased fertility and increased testosterone production and those with osteocalcin deficiencies Andrology, 1–7
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demonstrated the opposite (Oury et al., 2011). Although this study was conducted in mice, there is a human analogue to osteocalcin and its receptor, Gprc6a, and deficiency of this receptor was found in a recent study to be associated with infertility (Oury et al., 2013). Several recent human studies have sought to investigate the relationship between osteocalcin and testosterone production. Schwetz et al. correlated serum testosterone levels and carboxylated and uncarboxylated osteocalcin levels with sperm count in an infertile male population and found a statistically significant but weak correlation (Schwetz et al., 2013). Another study looked at bisphosphonate administration which lowers serum osteocalcin levels by ~40%, and found that it did not cause significant reductions in serum testosterone levels (Bolland et al., 2013). Other studies from Europe and Asia have shown promising, positive correlations at population levels with osteocalcin and testosterone (Kyvernitakis et al., 2013; Liao et al., 2013). Osteocalcin, a vitamin K-dependent protein, may be affected by medications such as warfarin, hydrochlorothiazide, bisphosphonates and calcitriol (Lian & Gundberg, 1988; Bolland et al., 2013). Based on these previous studies, it is possible that osteocalcin manipulation could provide a new opportunity for fertility and ED treatment, and perhaps for the treatment for hypogonadal sexual dysfunction in a way that does not adversely affect spermatogenesis (Smith & Saunders, 2011). The advantage of using the osteocalcin-testosterone regulatory loop as an approach to correct hypogonadism is that it may be done indirectly through improving bone health. However, a possible disadvantage is that targeting this loop may have unexpected consequences in other systems.
CONCLUSIONS Our understanding of the regulatory mechanisms of testosterone within the Leydig cell has greatly improved over the past two decades. Currently, pharmacologic treatments for hypogonadism are limited to those that act through the LH receptor, such as hCG, clomiphene, or aromatase inhibitors, or by bypassing endogenous production entirely through the exogenous administration of testosterone. Herein, we identified five future targets that might promote increased endogenous testosterone production through the Leydig cell instead of relying on exogenous testosterone administration. The common feature of each is that they do not rely upon the LH receptor. The potential approaches use small molecules, dietary supplements, or even FDA-approved medications. Although the data for these pharmacologic agents come primarily from animal studies, further study in human trials could prove promising and deserve further study.
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