Reviews of Reproduction (1998) 3, 141–144

Androgen receptor transactivation domain and control of spermatogenesis

Eu L. Yong, Farid Ghadessy, Qi Wang, Amparo Mifsud and Soon C. Ng

Department of Obstetrics and Gynaecology, National University of Singapore, Lower Kent Ridge Road, Singapore 119 074

Male steroids () are important for maintaining production and growth of the accessory , the gland. This article examines the role of the re- ceptor (AR) in the control of spermatogenesis and focusses on the N-terminal transactivation domain of the receptor, a poorly studied region that is essential for receptor function. This domain is of great interest because of its causative relationship to a fatal neuromuscular disease, spinal bulbar muscular atrophy (Kennedy’s syndrome). Genetic screening of the trans- activation domain of the AR of 153 patients presenting solely with defective spermato- genesis and , and of over 72 healthy fertile controls was performed. Up to 20% of infertile males have reduced androgenicity caused by an increase in length of a poly- morphic trinucleotide (CAG) repeat segment, encoding a polyglutamine tract, of the androgen receptor. The increased risk of male infertility associated with long CAG lengths is associated with reduced risk of prostate cancer. Conversely, short polyglutamine tracts are associated with increased risk of prostate cancer but a reduced risk of male infertility. Thus depressed sper- matogenesis and prostate cancer represent opposite ends of the spectrum of androgen receptor transactivation function. Improved understanding of androgen receptor action in these two important public health concerns could lead to rational and effective prevention and therapy.

Male sex steroids (androgens) sculpture the human embryo composed of three main functional domains: the trans- resulting in development of the male internal and external activation domain, the DNA-binding domain and the ligand- genitalia. At , a surge of androgens leads to the in- binding domain (Fig. 1). itiation of spermatogenesis and growth of accessory sex organs, Since the definition of the exon–intron boundaries of the including the prostate gland. Sperm production is highly de- androgen receptor gene, over 300 androgen receptor pendent on androgens (Zirkin et al., 1989) and it has long been (Gottlieb et al., 1998) have been reported in the ligand-binding suspected that suboptimal androgen action is one of the aetio- and DNA-binding domains, making it the most naturally mu- logical factors in idiopathic male infertility (Aiman and Griffin, tated transcription factor known. Most mutations are identified 1982). Since most infertile males have normal amounts of serum because they disrupt receptor function totally, causing com- androgen (Niechlag et al., 1979), it is possible that defective plete androgen insensitivity syndrome, wherein a healthy 46XY spermatogenesis could be the result of impaired androgen re- individual is born with external genitalia (Lim et al., sponsiveness. The cloning of the androgen receptor gene 1997). Mutations that do not completely disrupt androgen re- (Lubahn et al., 1988) and elucidation of its signal transduction ceptor function cause the partial syndrome, in which various pathway (Jenster et al., 1991) have provided new methods for degrees of ambiguous genitalia, including partial labioscrotal investigating the role of androgen receptor mutations in male fusion, hypospadias, bifid and gynaecomastia, occur infertility. All androgens transmit their multifaceted effects (Yong et al., 1998a). Mutations of the ligand-binding and DNA- through a single receptor: the androgen receptor (AR). The AR, binding domains that cause complete and partial androgen in- a member of the steroid receptor superfamily, is a nuclear tran- sensitivity syndromes have been reviewed comprehensively by scription factor that, when activated by androgens, switches on Quigley et al. (1995) and will not be discussed here. androgen-regulated that lead to spermatogenesis and Relatively few mutations have been reported in the trans- prostate gland development (for review see Quigley et al., 1995). activation domain because its large size (it comprises half of the Mutations of the androgen receptor are tolerated because the receptor) and high GC content make genetic screening difficult. receptor is not essential for life and, in males, mutations of The transactivation domain is essential for receptor function the X-linked receptor are expressed, providing a natural ‘knock- and its deletion leads to a non-functional in vitro out system’ for studying the phenotypic effects of the gene. The (Jenster et al., 1991). This domain is of particular interest be- AR gene encodes a receptor protein of apparent molecular cause of its causative relationship to a fatal neuromuscular mass 110–114 kDa including 910–919 amino acids. The full- disease, spinal bulbar muscular atrophy (Kennedy’s syn- length AR, like other steroid receptors, is a single polypeptide drome). This review will focus on the novel hypothesis that © 1998 Journals of Reproduction and Fertility 1359-6004/98 $12.50 Downloaded from Bioscientifica.com at 09/28/2021 07:02:11AM via free access 142 E. L. Yong et al.

patients have ≥ 40 CAG repeats compared with ≤ 35 in the normal population. The aetiology of trinucleotide expansions 5′ 123 4 5 6 7 8 3′ and the molecular mechanism of pathogenesis of neurological disease in SBMA is not known but is a subject of intense CAG GGC research. 1TAD DBD LBD 919 Patients with SBMA also have evidence of reduced andro- gen receptor function in the form of gynaecomastia, oligo- spermia (reduced sperm production) or (no sperm production), testicular atrophy and reduced fertility. Fig. 1. Simplified representation (not to scale) of the structure of the Investigators have also found an inverse relationship between androgen receptor gene and protein. The gene consists of eight polyglutamine length and androgen receptor function by exons that when translated result in a protein with three main do- expressing androgen receptor constructs of different CAG mains: the transactivation domain (TAD), the DNA-binding do- lengths in cell lines and measuring the androgen receptor main (DBD) and the ligand-binding domain (LBD). The TAD makes function by using reporter genes containing androgen response up half the receptor and has two polymorphic regions (orange elements. When androgen receptors with deleted polyglut- boxes) consisting of the trinucleotide repeats, (CAG) and (GGC) . n n amine tracts were assayed for their abilities to activate tran- scription of several different androgen receptor-responsive polymorphisms and mutations affecting the transactivation reporter genes, it was found that elimination of the tract in domain of the androgen receptor can cause subtle disruption both human and rat androgen receptors resulted in increased of receptor function and impairment of human spermato- transcriptional activation activity (Chamberlain et al., 1994). genesis without any associated defect in secondary sexual Progressive expansion of the CAG repeat in the human characteristics. The transactivation domain of the androgen re- androgen receptor caused a linear decrease in transactivation ceptor gene of 191 patients with male infertility due to defec- function but did not completely eliminate androgen receptor tive spermatogenesis and 101 healthy fertile controls was activity. A polyglutamine tract of normal size represses andro- screened for mutations. Genetic screening has revealed triplet gen receptor transactivation function and polyglutamine repeat polymorphisms that increase the risk of male infertility, expansion increases that effect. The relationship between and a point in the transactivation domain that causes polyglutamine tract and androgen receptor transactivation subtle impairment of receptor activity. was inverse and linear from 0 to 50 glutamines (Kazemi- Esfarjani et al., 1995). Triplet repeat polymorphisms in the transactivation domain Short CAG repeats associated with increased risk for prostate cancer Exon 1 of the androgen receptor gene, which encodes the transactivation domain, contains several regions consisting of Besides the pathological expansions of polyglutamine lengths repeats of three (Fig. 1). Two of these segments are found in SBMA, alleles of different sizes within the normal polymorphic, varying in size among normal individuals. The polymorphic range were found to be associated with the most interesting of these is the segment consisting of multiple androgen-dependent tumour, prostate cancer. The prevalence copies of the three nucleotides (CAG) that code for glutamine. of short CAG alleles (< 22 repeats) was highest (75%) in The CAG repeat segment contains an average of 21 ± 2 repeats African–American males with the highest risk of prostate with a range of 14–31 in the normal fertile male population (Tut cancer, intermediate (62%) in intermediate-risk non-Hispanic et al., 1997). Further downstream is an invariate stretch of eight whites, and lowest (49%) in Asians at very low risk of prostate (CCG)s, coding for eight proline residues. Closer to the DNA- cancer (Irvine et al., 1994). There was also an association of binding domain is the other polymorphic region consisting of short CAG repeats with increasing severity of disease. Thus, multiple repeats of codon GGC, which codes for the amino the median CAG repeat length was 25 for patients with stage A acid, glycine. This polyglycine tract consists of 24 GGC repeats prostate cancer, 22 for patients with stage B, 22 for patients and also differs among normal individuals. The crystal struc- with stage C, and 23 for patients presenting with stage D ture of the N-terminal domain is not known and this region is disease (Hardy et al., 1996). In another large case control the most variable among the steroid receptors. study of 587 cases of prostate cancer and 588 controls, it was The N-terminal transactivation domain has been the source found that men with ≤ 18 CAG repeats have a 1.5-fold higher of great interest in recent years because of the discovery that risk of prostate cancer than men with ≥ 26 repeats (Giovanucci expansion of the CAG repeat in the androgen receptor leads to et al., 1997). The shorter repeat sequence was also associated spinal bulbar muscular atrophy (SBMA), a fatal neuromuscular with extraprostatic extension and distant metastasis. In an disease (La Spada et al., 1991). Trinucleotide repeat expansions analysis of 301 cases, it was calculated that there was a are now implicated in many neuromuscular diseases (Mitas, 3% decreased risk in prostate cancer for each additional 1997), including , Huntington’s disease, CAG repeat (Stanford et al., 1997). Collectively, these data type 1, dentatorubral-pallidoluysian suggest that short CAG repeats have led to increased androgen atrophy, Machado-Joseph’s disease and Friedrich’s ataxia. In receptor androgenicity and abnormally high prostatic growth all these diseases, the polymorphic trinucleotide repeats were with a resultant earlier age of onset, increased tumour grade expanded well beyond the range that occurred in normal and increased risk of extra-prostatic extension in prostate unaffected persons. Thus, in the androgen receptor, SBMA cancer.

Downloaded from Bioscientifica.com at 09/28/2021 07:02:11AM via free access Androgen receptor and spermatogenesis 143

CAG repeats and defective spermatogenesis Polyglutamine length increase If short CAG lengths can lead to disease in androgen- dependent tissues, at the other end of the spectrum, long CAG repeats might in a similar fashion cause reduced androgen re- ceptor androgenicity and disease. Such a disease must be one that is extremely sensitive to reduction of androgen receptor function. In this light we decided to investigate spermato- genesis and CAG length. Spermatogenesis is very dependent Increasing androgenicity on large amounts of androgens. Testicular androgen concen- trations are about 50–100 times higher than they are in periph- eral blood and lowering of androgen concentrations causes Prostate Normal Defective SBMA severe disruption of sperm production (McLachlan et al., 1996). cancer spermatogenesis The lengths of the predicted polyglutamine and polyglycine re- peats in 153 patients with defective sperm production were compared with those of 72 normal controls of proven fertility by measuring the size of their CAG and GGC repeat segments Fig. 2. Schematic representation of the inverse relationship between (Tut et al., 1997). The polyglycine tract was less polymorphic the length of the polyglutamine tract and the function of the andro- and consisted of seven alleles. There were no significant dif- gen receptor. The greater the number of glutamines, the lesser the androgenicity, resulting in defective spermatogenesis. Expansion of ferences in the size of the polyglycine tracts between patients the polyglutamine tract beyond a threshold (vertical bar) leads to and controls. In contrast, the distribution of alleles encoding spinal bulbal muscular atrophy (SBMA), a condition in which de- the polyglutamine tract suggested that patients with reduced fective spermatogenesis and virilisation are also observed. sperm counts differed significantly, with respect to the preva- Conversely, short tracts are associated with increased androgenicity lence of glutamine number beyond 27, when compared with of the receptor resulting in increased risk of prostate cancer. normal fertile controls. Patients with ≥ 28 glutamines in their Normal receptor function lies in between that of prostate cancer androgen receptor had more than a fourfold increased risk of and defective spermatogenesis. reduced spermatogenesis. There was also a trend whereby the more severe the spermatogenic defect, the higher the propor- tion of patients with the longer polyglutamine alleles. The risk between number of glutamines and transregulatory activity of reduced spermatogenesis was halved with short repeat (Tut et al., 1997). The data indicate a direct relationship between lengths ≤ 23 Gln. For glutamine repeats ≤ 21, the odds ratio was the length of the polyglutamine tract and the risk of defective 0.44 suggesting that the shorter the length the lower the chance spermatogenesis that is attributable to the decreased functional of defective spermatogenesis. The data indicate an inverse re- competence of androgen receptor with longer glutamine tracts. lationship between the length of the polyglutamine tract in the Collectively, the evidence supports the hypothesis that the androgen receptor and the risk of reduced spermatogenesis glutamine repeat fine-tunes the balance between excess and (Fig. 2). Receptors with polyglutamine lengths of ≤ 23, the same deficient receptor function. The highly polymorphic nature of size that was found to give an excessive risk of prostate cancer the glutamine repeat would imply a subtle gradation of re- (Irvine et al., 1994), were associated with a reduced risk of male ceptor function among individuals, possibly allowing alleles infertility, suggesting that the greater androgenicity associated with evolutionary advantages to be rapidly selected and trans- with the shorter glutamine repeats could boost repli- mitted to future generations. This function of the androgen re- cation but with the long-term risk of overstimulating the ceptor polyglutamine length could be essential in view of the growth of prostatic tissue. changing amounts of steroid substances in the environment This study was extended to include over 300 patients, and and the demands of reproduction versus the requirements of comparing CAG lengths, in cases with male infertility, prostate other tissues affected by androgen. cancer and healthy fertile controls, using an automated It should be noted that the maximum polyglutamine length Genescan technique with internal size markers to minimize in these studies was 31, which is fewer than the 40 or more errors in assignation of repeat size (Yong et al., 1998b). It was found in SBMA patients. None of the patients with expanded found that CAG lengths fall into the following pattern: male CAG repeat segments exhibited any sign of neuromuscular dis- infertility > normal controls > prostate cancer. The increased ease. Moderate expansion of the polyglutamine tract (28–31 Gln) risk of male infertility associated with long CAG lengths was exerts a modulatory effect on usual androgen receptor func- associated with a reduced risk of prostate cancer. Conversely, tion, whereas expansion beyond a threshold (≥ 40 Gln) is likely short polyglutamine tracts are associated with increased risk of to trigger a separate process that is neurotoxic (Fig. 2). It is esti- prostate cancer but a reduced risk of male infertility. Thus, it mated that between 10 and 20% of patients with male infertility appears that extremes of variation in androgen receptor poly- could have reduced androgen receptor function as a result of glutamine length, associated with high or low receptor activity, long polyglutamine tracts. in the normal population can lead to disease. Gene transfer experiments, in which androgen receptor con- Mutational hot spot in the transactivation domain structs with 15, 20 and 31 glutamine repeats, and a luciferase reporter gene with an androgen-response-element promoter, Besides the CAG repeat, genetic screening of the trans- were co-expressed in vitro, confirmed an inverse relationship activation domain in patients with male infertility revealed

Downloaded from Bioscientifica.com at 09/28/2021 07:02:11AM via free access 144 E. L. Yong et al. three different single strand conformation polymorphisms and *Irvine RA, Yu MC, Ross RK and Coetzee GA (1994) The CAG and GGC sequencing of the mutant fragments revealed three G→A tran- microsatellites of the androgen receptor gene are in linkage dis- equilibrium in men with prostate cancer Cancer Research 54 2861–2864 sitions in codons 210, 211 and 214 (Wang et al., 1998b). The first Jenster G, Van der Korput HAGM, Van Vroonhoven C, Van der Kwast TH, two mutations were polymorphisms and the transition in Trapman J and Brinkmann AO (1991) Domains of the human androgen codon 211 was related to ethnic origin, occurring in 10–15% of receptor involved in steroid binding, transcriptional activation, and sub- Indian or Middle-Eastern subjects, but not in ethnic Chinese cellular localisation Molecular Endocrinology 5 1396–1404 who comprise the majority of the subjects. These two poly- Kazemi-Esfarjani P, Trifiro Mav and Pinsky L (1995) Evidence for a re- pressive function of the long polyglutamine tract in the human androgen morphisms could therefore be useful in tracking racial origins. receptor: possible pathogenetic relevance for the (CAG)n-expanded neur- The third mutation resulted in a non-conservative glycine to onopathies Human Molecular Genetics 4 523–527 arginine substitution at codon 214 (G214R) and was associated La Spada AR, Wilson EM, Lubahn DB, Harding AE and Fischbeck KH with about 20% lower transactivation capacity than the wild (1991) Androgen receptor gene mutations in X-linked spinal and bulbar muscular atrophy Nature 352 77–79 type. The G214R mutation was found in a patient with male Lim J, Farid J, Ghadessy FJ and Yong EL (1997) A novel splice site mutation infertility as well as in a fertile control. Thus, although a poss- in the androgen receptor gene results in exon skipping and a non- ible pathogenic mutation was uncovered, mutations of the non- functional protein Molecular and Cellular Endocrinology 131 205–210 polymorphic portions of the transactivation domain do not Lubahn DB, Joseph DR, Sullivan PM, Willard HF, French FS and Wilson EM appear to have a major role in the aetiology of idiopathic male (1988) Cloning of human androgen receptor complementary DNA and localization to the X Science 240 327–330 infertility. McLachlan RI, Wreford NG, O’Donnell L, de Kretser DM and Robertson DM (1996) The endocrine regulation of spermatogenesis: independent roles for and FSH Journal of Endocrinology 148 Conclusion 1–9 Mitas M (1997) Trinucleotide repeats associated with human disease Nucleic These naturally occurring mutations and polymorphisms indi- Acids Research 25 2245–2254 cate that the transactivation domain has an important function Nieschlag E, Hertle L, Fischedick A and Behre HM (1979) Endocrine tes- in modifying androgen receptor activity. A full understanding ticular function in vivo and in vitro in infertile men Acta Endocrinologica of the function of this domain would lead to better understand- (Copenhagen) 90 544–551 ing of the aetiology of male infertility and prostate cancer. *Quigley CA, De Bellis A, Marschke KB, El Awady MK, Wilson EM and French FS (1995) Androgen receptor defects: historical, clinical and mol- A challenge for the future is to determine the mechanisms ecular perspectives Endocrine Reviews 16 271–321 by which this is achieved and to devise rational therapeutic Stanford JL, Just JJ, Gibbs M, Wicklund KG, Neal CL, Blumenstein BA and measures for these distressing conditions. Ostrander EA (1997) Polymorphic repeats in the androgen receptor gene: molecular markers of prostate cancer risk Cancer Research 57 1194–1198 *Tut TG, Ghadessy F, Trifiro MA, Pinsky L and Yong EL (1997) Long poly- References glutamine tracts in the androgen receptor are associated with reduced transactivation, defective sperm production and male infertility Journal of Key references are identified by asterisks. Clinical Endocrinology and Metabolism 82 3777–3782 Aiman J and Griffin JE (1982) The frequency of androgen receptor deficiency *Wang Q, Ghadessy FJ and Yong EL (1998) Mutations of the transactivation in infertile men Journal of Clinical Endocrinology and Metabolism 54 domain of the androgen receptor gene in patients with defective sper- 725–732 matogenesis Clinical Genetics (in press) Chamberlain NL, Driver ED and Miesfeld RL (1994) The length and location Yong EL, Tut TG, Ghadessy FJ, Prins G and Ratnam SS (1998a) Partial of CAG trinucleotide repeats in the androgen receptor N-terminal domain androgen insensitivity and correlations with the predicted three di- affect transactivation function Nucleic Acids Research 122 3181–3186 mensional structure of the androgen receptor ligand-binding domain Giovannucci E, Stampfer MJ, Krithivas K et al. (1997) The CAG repeat Molecular and Cellular Endocrinology 137 41–50 within the androgen receptor gene and its relationship to prostate cancer *Yong EL, Liu PP, Sim CS, Vasan SS, Lim PHC and Ng SC (1998b) The CAG Proceedings of the National Academy of Sciences, USA 94 3320–3323 repeat in the androgen receptor gene is associated with increased risk for Gottlieb B, Lehvaslaiho H, Beitel LK, Lumbroso R, Pinsky L and Trifiro M either male infertility or prostate cancer Proceedings of the 80th Annual (1998) The androgen receptor gene mutations database Nucleic Acids Meeting of the Endocrine Society (USA) Abstract OR43-3, p.108 Research 26 234–238 Zirkin BR, Santulli R, Awoniyo CA and Ewing LL (1989) Maintenance of Hardy DO, Scher HI, Bogenreider T et al. (1996) Androgen receptor CAG advanced spermatogenic cells in the adult rat testis: quantitative relation- repeat lengths in prostate cancer: correlation with age of onset Journal of ship to testosterone concentration within the testis Endocrinology 124 Clinical Endocrinology and Metabolism 81 4400–4405 3043–3049

Downloaded from Bioscientifica.com at 09/28/2021 07:02:11AM via free access