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Developmental aspects of androgen action
Molecular and Cellular Endocrinology 185 (2001) 33 – 41 www.elsevier.com/locate/mce
Developmental aspects of androgen action I.A. Hughes a,*, H.N. Lim a, H. Martin a, N.P. Mongan a, L. Dovey a, S.F. Ahmed b, J.R. Hawkins c a
Department of Paediatrics, Uni6ersity of Cambridge, Addenbrooke’s Hospital, Box 116, Cambridge CB2 2QQ, UK b Department of Child Health, Royal Hospital for Sick Children, Glasgow G3 8SJ, UK c Incyte Genomics, Cambridge Science Park, Cambridge CB4 0WA, UK
Abstract The formation of a testis from the indifferent gonad is the prelude to sequential steps in male sex differentiation orchestrated by time-dependent androgen biosynthesis and action. Information about the cellular and molecular mechanisms of androgen action can be obtained by the study of disorders of sex differentiation in males. The pivotal role of the androgen receptor as a ligand-induced transcription factor is emphasised and preliminary studies are described which attempt to identify developmentally regulated androgen-responsive genes. That androgen action can be modulated by gene polymorphisms is illustrated by the influence of an androgen receptor polyglutamine repeat in the multi-factorial causation of less severe forms of male undermasculinisation. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Sex differentiation; Androgen receptor; Co-regulators; Androgen-responsive genes; Polyglutamine repeats
1. Introduction The determination of a testis from the bipotential, indifferent gonad is critical in the male for subsequent differentiation of the internal and external genitalia. The factors involved in testis determination have been the subject of several recent reviews (Goodfellow and Lovell-Badge, 1993; Graves, 1998; Lim et al., 1998; Wylie, 2000; Quigley, 2001). The developmental aspects of androgen action are thus the consequence of differentiated Leydig cells synthesising sufficient quantities of androgen to act on target tissues during early fetal life. Knowledge about this process can be gleaned indirectly by the study of disorders of sex differentiation associated with insensitivity to the action of androgens, the syndromes of androgen insensitivity (AIS).
2. Aspects of embryology Fig. 1 depicts the major events in male fetal developmental in a temporal fashion. The first event following * Corresponding author. Tel.: + 44-1223-336885; fax: + 44-1223336996. E-mail address: [email protected] (I.A. Hughes).
testis determination is the regression of Mullerian ducts under the influence of anti-Mullerian hormone (AMH), a member of the transforming growth factor b family and secreted by the Sertoli cells. This hormone signals through binding to an AMH Type II receptor expressed in the mesenchyme of Mullerian ducts to effect regression of these structural anlage by 10 weeks of fetal age (Josso et al., 1997; Belville et al., 1999; Allard et al., 2000). The onset of testosterone synthesis is initially gonadotrophin-independent before becoming placental hCG-dependent and thereafter under the control of fetal pituitary luteinizing hormone. Concentrations of testosterone in fetal serum increase to within the adult male range and coincide with the period of Wolffian duct stabilisation in the male (Voutilainen, 1992). Fetal testes during the second trimester show histological evidence of Leydig or interstitial cell hyperplasia and immunohistochemistry reveals the expression of steroidogenic enzymes for testosterone synthesis and the androgen receptor (AR) for androgen action (Codesal et al., 1990; Murray et al., 2000). The role of androgens in stabilising Wolffian ducts to differentiate as the internal male genital ducts (vas deferens, epididymis and seminal vesicles) is based primarily on the results of fetal castration experiments in rabbits (Jost,
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1953). Unilateral castration is associated with destabilisation of Wolffian ducts which can be corrected by unilateral local application of testosterone. The preservation of Wolffian ducts in some patients with a defect in androgen biosynthesis or in responsiveness to normal androgen concentrations suggests that trophic factors in addition to androgens may play a role in Wolffian duct stabilisation (Boehmer et al., 1999; Gupta, 1996; Hodapp and Hughes 1999). The absence of androgens, however, appears to be the sole explanation for Wolffian duct regression in the female. Androgens also play a key developmental regulatory role in the differentiation of the male external genitalia. The external genital anlage are indifferent until 8 weeks of gestation when, in the male under the influence primarily of dihydrotestosterone (DHT), the genital tubercle elongates to form the penis, the urethral folds fuse to develop the penile urethra and labioscrotal folds fuse posteriorly to form the scrotum. The evidence for DHT-dependence is based on expression of the 5a-reductase Type II enzyme in these tissues (Thigpen et al., 1993) and the failure of fetal external genital development in males who lack this enzyme (Wilson et al., 1993). Development of the prostate gland is also dependent on DHT. Continued growth of the penis in later fetal life requires testosterone, synthesised now by the testis under the control of LH secretion by the fetal pituitary. Micropenis may be the consequence of prenatal anterior pituitary deficiency. Descent of the testes into the scrotum by the end of human gestation is the final step in the regulation of male fetal development. Migration of the testis from the lower pole of the kidney into the scrotum is a two-stage process of transabdominal and inguinoscrotal descent (Hutson and Beasley, 1992). Degeneration of the cranial suspensory ligament which attaches the testis to the lower pole of the kidney is part androgendependent (Emmen et al., 1998). The gubernaculum develops from the caudal suspensory ligament and is
critical for testicular descent. Its development is not androgen dependent and in the mouse, appears to be controlled by insulin-like factor 3 (insl3). Insl3 − / − mice have intra-abdominal testes and poorly formed gubernacula (Zimmermann et al., 1999; Nef and Parada, 1999). Recent studies in boys with bilateral cryptorchidism indicate that mutations in the human INSL3 gene associated with this disorder are rare (Koskimies et al., 2000; Tomboc et al., 2000; Lim et al., 2000). The final stage of inguinoscrotal descent also appears to be androgen-dependent, based on evidence of undescended testes in association with hypogonadotrophic hypogonadism and the siting of testes in syndromes associated with androgen insensitivity (Lim et al., 2001c).
3. Mechanism of androgen action Male sex development is co-ordinated by ligand activation of the AR, a nuclear transcription factor which controls androgen-dependent gene expression. The AR is ubiquitously expressed, including tissues which differentiate as the internal and external male genitalia. Current evidence indicates that a single AR binds all androgens intracellularly in target cells (Fig. 2). A single copy gene on chromosome Xq11–12 encodes the AR. Circulating androgens dissociate from carrier proteins, specifically sex hormone binding globulin, prior to diffusing into target cells and binding to the intracellular AR. The unliganded receptor is an inactive oligomer complexed to heat shock proteins (eg Hsp90, Hsp70) and located in the cytoplasm. The oligomeric complex dissociates when bound to hormone, the receptor undergoes a conformational change while transporting to the nucleus and binding as a homodimer to DNA hormone response elements (White and Parker, 1998). Activation of gene transcription via these specific hormone response elements is also a feature of the
Fig. 1. Embryologic events in male sex differentiation depicted in temporal fashion. The line depicts the increase in fetal serum testosterone concentrations.
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Fig. 2. Schematic of androgen receptor action.
related receptors for the glucocorticoid, mineralocorticoid and progestogen classes of hormones. This subfamily of nuclear receptors is but a small component of a large super-family of nuclear receptors which include receptors for other classes of steroids (oestrogens, vitamins D), thyroid hormones retinoids, thiazolidenediones (PPARg receptor) and a large group of receptors whose ligands are not known (orphan receptors). In common with other nuclear receptors, the AR is characterised by three functional domains involved in the regulation of transcription and binding to DNA and hormones (Fig. 3). A large N-terminal domain is the least conserved amongst the nuclear receptor family and contains an activation function (AF-1) region which is autonomously involved in gene transactivation. A unique feature of the AR is a polymorphic glutamine region in the N-terminus which is the result of a variable number of CAG repeats toward the 5% end of the AR gene first exon. There is evidence that variations in the length of the polyglutamine tract have effects on AR transcriptional efficiency (see later). The central DNA-binding domain is the most conserved region; the C-terminal part of the receptor contains the ligand-binding domain within which is a second activation function region (AF-2), also involved in gene transactivation (Jenster et al., 1991). Other functions mediated by the C-terminus include heat shock protein interactions, dimerisation, nuclear localisation signalling as well as ligand binding. The activation function (AF) regions interact with an intermediary group of proteins in order that transcriptional regulation of specific genes via the general transcriptional machinery be initiated (Raeder, 1996). This group of proteins, termed co-regulators, form protein:protein interactions in a ligand-dependent manner to either increase (co-activator) or decrease (co-repressor) gene transcription (McKenna et al., 1999; Robyr et al., 2000). Fig. 4 illustrates the interaction of
ligand-bound AR homodimers in a multiprotein complex with SRC-1, a representative member of the nuclear receptor coregulator family (Onate et al., 1995). The function of AF-2 is ligand-dependent and is located within one of the alpha-helices (helix 12) which binds to receptor-interacting motifs (LXXLL; L is leucine, X is any amino acid) of co-regulators (Darimont et al., 1998). This model appears to be generic to the mechanism of action of all ligand-activated nuclear receptors. In addition, the AR is unique in displaying constitutive activity in vitro when experiments involving deletion of the ligand-binding domain were performed (Jenster et al., 1991; Zhan et al., 1994). This suggests a critical role for the ligand-independent AF-1 region in gene transactivation in the case of the AR. In this instance, the interaction with the SRC-1 co-regulator appears not to be via LXXLL motifs but through interaction with a conserved, glutamine-rich region in the C-terminal region of SRC-1 (Bevan et al., 1999). Also depicted in Fig. 4 as part of the multiprotein complex is SRA (steroid receptor RNA activator) which is selective for the AF1 region of steroid receptors and uniquely functions as an endogenous RNA transcript (Lanz et al., 1999). Another co-regulator which appears to be relatively specific for interaction with the ligand-binding domain of the AR is ARA70 (Yeh and Chang, 1996; Gao et al., 1999).
Fig. 3. Structure – function characteristics of the androgen receptor. The letters denote the eight exons of the AR gene and the arrows delineate the three main functional domains of the AR. Glutamine (Gln) and glycine (Gly) are polymorphic regions in the N-terminus. AF-1, activation function 1; AF-2, activation function 2.
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Fig. 4. Schematic of ligand-bound AR interacting with co-regulator proteins. p160/SRC-1 (steroid receptor coactivator 1), CBP (CREBbinding protein), pCAF (CBP-associated factor) are representative examples of the large superfamily of coregulators. SRA (steroid receptor RNA activator) functions as an RNA transcript. DHT, dihydrotestosterone.
Androgen action in target cells is mediated via a complex pathway comprising multiple steps and interactions involving ligand, protein:protein and protein:DNA binding. Information about the critical steps in this pathway can be gained by studying XY sex reversed patients whose phenotype is the result of insensitivity to action of normal or increased concentrations of circulating androgens.
4. Syndrome of androgen insensitivity The androgen insensitivity syndromes (AIS) are defined by the complete (CAIS) or partial (PAIS) absence of signs of androgen responsiveness in genetic (XY) males with normal testis determination and androgen biosynthesis (Quigley et al., 1995; Ahmed et al., 2000a). AIS is the clinical paradigm of a hormone resistance syndrome of which there are now numerous examples relating to both nuclear receptor and cell membrane receptor-related cell signalling systems (Jameson, 1999). Characteristic features of CAIS include complete XY sex reversal in the form of a normal external female phenotype apart from the absence of, or scanty growth of, pubic and axillary hair. Internal female genital ducts are absent as the result of the action of AMH secreted by the testes; there is a blindending, shortened vagina whose development is stabilised independent of AMH or oestrogen effects. There may be variable descent of the testes but in CAIS, they are typically located in the labia. When some degree of masculinisation has occurred, such as clitoromegaly, this defines the partial form of AIS (PAIS). The external phenotype in PAIS can be variable but a common manifestation is micropenis, severe perineoscrotal hypospadias and a bifid scrotum which may or may not contain testes. A number of classification systems exist which attempt to quantify the degree of under-masculinisation (Quigley et al., 1995; Cameron et al., 1997; Ahmed et al., 2000b). A
Fig. 5. Cambridge Intersex Database. Diagnostic categories represented in the sample collection.
form of PAIS is also recognised where male factor infertility is the sole manifestation in an otherwise normally masculinised male (Giwercman et al., 2001). Numerous mutations distributed throughout the AR gene have now been reported and they are detailed on an international database (Gottlieb et al., 1998http// www.mcgill.ca/androgendb/). The Cambridge Intersex database was established to gather clinical, biochemical, histological and molecular information on XY intersex cases. As a result of a successful collaboration with numerous clinicians in the UK and mainland Europe, data is now available on more than 1000 cases. Fig. 5 illustrates the diagnostic categories, defined primarily on clinical criteria. There is a bias towards AIS because of the research interests of the Department, but the database has a representative number for other causes of XY sex reversal such as gonadal dysgenesis syndromes and defects in androgen biosynthesis. Mutations in the AR gene can be identified in the majority of patients with clinical and biochemical evidence of CAIS. In contrast, only a minority of patients with PAIS as defined by under-masculinisation in the absence of any abnormality in androgen synthesis, have mutations identified in the AR gene. A representative sample of AR mutations from the Cambridge Intersex database is shown in Fig. 6. The mutations are distributed throughout the gene and comprise a mix of missense, nonsense, deletions and insertions. There is a preponderance of mutations which affect the ligand
Fig. 6. Mutation analysis of the AR gene in AIS from samples in the Cambridge Intersex Database. Mutations are located according to exons A-H and type indicated by the symbols.
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binding domain of the AR. Functional analysis of these mutations provides indirect evidence about critical regions of the AR in support of the recently reported crystal structure of the AR ligand binding domain (Matias et al., 2000). The crystal structures of several other nuclear receptor ligand binding domains have also been determined but not yet for a complete receptor (Moras and Gronemeyer, 1998; White and Parker, 1998). They have in common 12a – helices arranged in anti-parallel fashion in three layers in the form of a sandwich fold. Helix 12 is the most C-terminal and in the presence of ligand becomes realigned to form a hydrophobic cleft where the LXXLL motifs of certain co-regulators bind. In the case of the AR, the critical regions for ligand binding can be discerned by studying the functional consequences in vivo and in vitro of missense mutations affecting the ligand binding domain. For example, using homology modelling based on the known crystal structure of the progesterone receptor, arginine 779 is critical to ligand binding and subsequent transactivation whereas a histidine 874 alanine substitution has only a minimal effect on androgen binding (Poujol et al., 2000). Mutations in the AR ligand binding domain appear to cluster in halices H4 and H5 which is the region involved in ligand binding.
5. Beyond the androgen receptor CAIS is a clearly defined disorder based on clinical features, biochemical evidence of LH-induced androgen hypersecretion and Leydig cell hyperplasia. Typically, there is absent specific AR binding in genital skin fibroblasts and a deleterious mutation of the AR gene is usually identified. The positive yield in identifying mutations in the AR gene in the PAIS form is much less, even when all the evidence points to androgen resistance causing the phenotype. This raises the possibility that defects extraneous to the AR may also manifest as androgen resistance. Two areas worthy of exploration are abnormalities in the family of nuclear co-regulator proteins and secondly, a defect in a gene responsive to androgen-induced AR transcriptional activation.
5.1. Co-regulator dysfunction Compelling evidence for the role of co-regulators mediating the optimal effect of ligand-activated nuclear receptors came from the results of disrupting the SRC-1 gene in mice (Xu et al., 1998). Target organs which are sex hormone dependent such as the mammary gland, uterus, prostate and testis showed reduced growth responsiveness in vivo to sex steroid administration compared with mice containing an intact SRC-1 gene. Also observed was over-expression of a related co-regulator, TIF2 (also known as SRC-2), perhaps in part compen-
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sation for the loss of SRC-1 activity. Further studies in the null mutant mice showed evidence also for thyroid hormone resistance based on elevated serum TSH concentrations in the face of serum thyroxine concentrations higher than in the serum of wild-type mice (Weiss et al., 1999). Hypothyroidism was induced in normal and mutant mice using a diet low in iodine and containing propylthiouracid. Serum TSH concentrations were elevated to a similar degree but the negative feedback regulation of TSH by exogenous thyroxine administration was much less sensitive in the SRC-1 knock-out mice. These animal studies by analogy suggest that some human nuclear hormone receptor resistant states may be caused by co-factor dysfunction in cases where the cognate receptor is intact. Only a few studies of co-regulators in human hormone resistance syndromes are reported to date. The report of two sisters with clinical and biochemical resistance to glucocorticoids, mineralocorticoids, and androgens but not thyroid hormones was postulated to be caused by a co-activator defect. However, no analyses were performed on any known co-activator (New et al., 1999). More specifically, a recent case report described a patient with classical features of CAIS in whom the AR gene was normal (Adachi et al., 2000). In a series of experiments using truncated and chimeric nuclear receptor constructs to assess the functional interactions between the receptors in genital skin fibroblasts and several co-activator proteins, the transmission of a transactivation signal from the AF-1 region was impaired in fibroblasts from the patient. An unknown protein corresponding to a 90 kD band which interacted with the AF-1 region of the AR was present only in fibroblasts from controls. Clearly, while further studies are required to determine the nature of this protein which appears to be a specific co-activator for the AR, the observation does raise the possibility of a novel explanation for some forms of androgen resistance (Hughes, 2000). One co-activator shown to be relatively AR-specific in human prostate cells is ARA70 which is highly homologous to the thyroid expressed RET-fused gene and displays ligand- dependent interaction with the AR (Yeh and Chang, 1996). In co-transfection assays, ARA70 acts as a relatively weak coactivator (Gao et al., 1999). We postulated that any dysfunction of ARA70 in humans could result in a milder degree of undermasculinisation compared with the phenotype produced by a deleterious AR gene mutation. The ARA70 cDNA was screened by single stranded conformational polymorphisms and heteroduplex analysis in a group of XY patients with varying degrees undermasculinisation in whom defects in the AR had been excluded (Lim et al., 2001a). No mutations which changed the amino acid sequence of ARA70 were identified or any other type of mutation. The large family of
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Fig. 7. Suppression subtractive hybridisation: differential screening of clones to determine preliminary expression profiles in cDNA populations from genital skin fibroblasts: (a) transcript in untreated cDNA population (a putative androgen repressed sequence; (b) transcript in treated cDNA population (a putative androgen induced sequence); (c) transcript in both cDNA populations, but at differing levels of transcription.
nuclear co-regulator proteins play a significant role in transcriptional regulation in a combinatorial and ligand-dependent manner. Whether the action of any one member is so specific as to cause a hormone resistance state when dysfunctional has yet to be determined in humans.
5.2. Androgen-responsi6e genes Little is known about target genes which respond to androgens, either developmentally or postnatally at the time of puberty. Yet there is an abundance of biological actions of androgens which must depend on the up-regulation of target specific genes and protein expression. Some are well characterised such as the prostate-specific proteins, probasin and prostate specific antigen (Rennie et al., 1993; Young et al., 1991). We have utilised the expression-based cloning method, Suppression Subtractive Hybridisation (SSH), (Diatchenko et al., 1996) to generate cDNA libraries enriched for androgen-regulated transcripts. To perform SSH, two cDNA populations are synthesised and PCR strategies utilised to selectively amplify sequences that are unique to either population. The cDNA populations were synthesised from genital skin fibroblasts which express high levels of the AR. Cells were cultured under carefully defined growth conditions, to limit the introduction of androgens from standard culture medium and separately treated with dihydrotestosterone and the synthetic androgen, mibolerone. After
SSH, the resultant cDNA libraries are normalised and enriched for androgen-induced and repressed sequences from the treated and untreated cultures, respectively. To determine the expression profile of individual sequences within the libraries, all sequences were cloned in bacterial hosts and arrayed for radioactive hybridisation with cDNA from treated and untreated cultures, both pre- and post-subtraction (Fig. 7). This allowed false positive clones to be removed and preliminary expression profiles to be determined. The expression profile of individual candidate clones was confirmed by Southern and Northern hybridisation prior to sequencing and further analysis (Fig. 8). Using these techniques, we have isolated a number of transcripts that demonstrate differential expression profiles between androgen treated and untreated fibroblast cultures (Fig. 8, Clone 1). In addition to sequences that are unique to either the treated or untreated cDNA populations, we have also isolated sequences present in both populations but at differing concentrations (Fig. 8, Clone 2). This suggests that dose sensitive transcription effects may also feature in androgen regulation of gene activity. The use of SSH and additional expression-based cloning techniques allows a catalogue of differentially and quantitatively expressed sequences that demonstrate androgen regulation to be compiled. The specific developmental pathways regulated by androgens may be better characterised on identification and isolation of such expressed sequences.
I.A. Hughes et al. / Molecular and Cellular Endocrinology 185 (2001) 33–41
Fig. 8. Southern hybridisation of two candidate clones to cDNA target DNA synthesised from androgen treated and untreated fibroblasts. Arrow indicates differentially expressed sequence in Clone 1 hybridisation. Clone 2 demonstrates quantitative differences in expression.
6. Factors which modulate androgen action The importance of optimal androgen production and action during the critical developmental phase of male sex differentiation has been emphasised. There is increasing evidence that polymorphic variants of gene products may modulate hormone function. In the case of androgens, a variable number of glutamine repeats in the N-terminal region of the AR is associated with disorders affecting the male reproductive system. Thus a hyper-expanded AR polyglutamine tract which leads to the neurodegenerative disorder, spinal and bulbar muscular atrophy, also results in androgen dysfunction manifest as mild androgen insensitivity, testicular atrophy and decreased spermatogenesis (La Spada et al., 1991). However, variations in the number of glutamines within the normal range (11– 31 repeats) are associated with male reproductive disorders such as decreased
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spermatogenesis in otherwise normal males (Tut et al., 1997; Dowsing et al., 1999). In vitro studies indicate that an AR containing a longer polyglutamine tract is transcriptionally less active; indeed, the opposite in vitro observation is the case with shorter tracts. There is conflicting evidence that a shorter AR glutamine repeat may be not only a risk factor for developing prostate cancer but also influence the age of onset and response to treatment involving endocrine ablation (Stanford et al., 1997; Bratt et al., 1999; Correa-Cerro et al., 1999). We have previously demonstrated that longer polyglutamine repeats within the normal range are associated with varying degrees of undermasculiniation of unknown cause (Lim et al., 2000) (Fig. 9). The estimated increase in the odds ratio for each additional repeat was 9.1%. In a subsequent study of a larger number of males with abnormal genital development, we found evidence that a longer AR glutamine repeat may contribute to the cause of genital maldevelopment, particularly with less severe genital abnormalities (Lim et al., 2001b). Furthermore, there was a strong association between decreasing severity of the genital malformations and an increasing proportion with a multifactorial aetiology. On the basis of these findings, a model for the involvement of the AR polyglutamine length in genital abnormalities is proposed (Fig. 10). It suggests that the relative impairment of AR transcriptional activity consequent upon a longer repeat is a contributory cause in a multifactorial fashion to the syndrome of moderate undermasculinisation. In contrast, when hormone resistance is absolute (complete sex reversal as in CAIS), the contribution of the longer AR glutamine repeat to the genital malformation is negligible. Further studies of larger patient populations analysing multiple genes involved in the control of androgen production and action will contribute to a better understanding of the developmental aspects of androgen action in the human.
Fig. 9. Distribution of AR glutamine repeats in undermasculinised subjects compared with controls.
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Fig. 10. A model incorporating the effect of an AR polymorphism on the aetiology of genital abnormalities. The influence of a longer glutamine repeat is greater when multi-factorial causes lead to moderate genital abnormalities.
Acknowledgements The support of the Birth Defects Foundation, European Community, Sir Halley Stewart Trust and the Cambridge Children’s Kidney Care Fund for some of the studies described in this paper is gratefully acknowledged.
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I.A. Hughes et al. / Molecular and Cellular Endocrinology 185 (2001) 33–41 the human androgen receptor involved in steroid binding, transcriptional activation, and subcellular localization. Mol. Endocrinol. S., 1396 – 1404. Josso, N., Picard, J.Y., Imbeaud, S., di Clemente, N., Ray, R., 1997. Clinical aspects and molecular genetics of the persistent Mullerian duct syndrome. Clin. Endocrinol. 47, 137 –144. Jost, A., 1953. Problems of fetal endocrinology: the gonadal and hypophyseal hormones. Progr. Horm. Res. 8, 379 –418. Koskimies, P., Virtanen, H., Lindstrom, M., et al., 2000. A common polymorphism in the human relaxin-like factor (RLF) gene: no relationship with cryptorchidism. Pediatr. Res. 47, 538 –541. Lanz, R.B., McKenna, N.J., Onate, S.A., et al., 1999. A steroid receptor coactivator, SRA, functions as an RNA and is present in an SRC-1 complex. Cell 97, 17 –27. La Spada, A.R., Wilson, E.M., Lubahn, D.B., Harding, A.E., Fishbeck, K.H., 1991. Androgen receptor gene mutations in X-linked spinal and bulber muscular atrophy. Nature 352, 77 –79. Lim, H.N., Chen, H., McBride, S., et al., 2000. Longer polyglutamine tracts in the androgen receptor are associated with moderate to severe undermasculinized genitalia in XY males. Hum. Mol. Genet. 9, 829 – 834. Lim, H.N., Freestone, S.H., Romero, D., Kwok, C., Hughes, I.A., Hawkins, J.R., 1998. Candidate genes in complete and partial XY sex reversal: mutation analysis of SRY, SRY-related genes and FTZ-F1. Mol. Cell. Endocrinol. 140, 51 – 58. Lim, H.N., Hawkins, J.R., Hughes, I.A., 2001a. Genetic evidence to exclude the androgen receptor co-factor, ARA 70 (NCOA4) as a candidate gene for the causation of undermasculinised genitalia. Clin. Genet. (in press). Lim, H.N., Nixon, R.M., Chen, H., Hughes, I.A., Hawkins, J.R., 2001b. Evidence that longer androgen receptor polyglutamine repeats are a causal factor for genital abnormalities. J. Clin. Endocrinol. Metab. (in press). Lim, H.N., Rajpert-De Meyts, E., Skakkebaek, N.E., Hawkins J.R., Hughes I.A., 2001c. Genetic analysis of the INSL3 gene in patients with maldescent of the testis. Eur. J. Endocrinol. (in press). Matias, P.M., Donner, P., Coelho, R., et al., 2000. Structural evidence for ligand specifically in the binding domain of the human androgen receptor. J. Biol. Chem. 275, 26 164 –26 171. McKenna, N.J., Lanz, R.B., O’Malley, B.W., 1999. Nuclear receptor coregulators: cellular and molecular biology. Endocr. Rev. 20, 321 – 344. Moras, D., Gronemeyer, H., 1998. The nuclear receptor ligand-binding domain: structure and function. Curr. Opin. Cell Biol. 10, 384 – 391. Murray, T.J., Fowler, P.A., Abramovich, D.R., Haites, N., Lea, R.G., 2000. Human fetal testis: second trimester proliferative and steroidogenic capacities. J. Clin. Endocrinol. Metab. 85, 4812 – 4817. Nef, S., Parada, L.F., 1999. Cryptorchidism in mice mutant for Insl3. Nat. Genet. 22, 295 –299. New, M.I., Nimkarn, S., Brandon, D.D., et al., 1999. Resistance to several steroids in two sisters. J. Clin. Endocrinol. Metab. 84, 4454 – 4464. Onate, S.A., Tsai, S.Y., Tsai, M.J., O’Malley, B.W., 1995. Sequence and characterization of a coactivator for the steroid hormone
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receptor superfamily. Science 270, 1354 – 1357. Poujol, N., Wurtz, J.-M., Tahiri, B., 2000. Specific recognition of androgens by their nuclear receptors. J. Biol. Chem. 275, 24 022 – 24 031. Quigley, C.A., 2001. Genetic basis of sex determination and sex differentiation. In: DeGroot, L.J., Jameson, J.L. (Eds.), Endocrinology, 4th. W.B. Saunders, London, pp. 1926 – 1949. Quigley, C.A., De Bellis, A., Marschke, K.B., El-Awady, M.K., Wilson, E.M., French, F.S., 1995. Androgen receptor defects: historical, clinical and molecular perspectives. Endocr. Rev. 16, 271 – 321. Raeder, R.G., 1996. The role of general initiation factors in transcription by RNA polymerase II. Trends. Biosci. 21, 327 – 335. Rennie, P.S., Bruchovsky, N., Leco, K.J., et al., 1993. Characterisation of two cis-acting DNA elements involved in the androgen regulation of the probasin gene. Mol. Endocrinol. 7, 23 –36. Robyr, D., Wolffe, A.P., Wahli, W., 2000. Nuclear hormone receptor coregulators in action: diversity for shared tasks. Mol. Endocrinol. 14, 329 – 347. Stanford, J.L., Just, J.J., Gibbs, M., et al., 1997. Polymorphic repeats in the androgen receptor gene: molecular markers of prostate cancer risk. Cancer Res. 57, 1194 – 1198. Thigpen, A.E., Silver, R.I., Guileyardo, J.M., et al., 1993. Tissue distribution and ontogeny of steroid 5a-reductase isozyme expression. J. Clin. Invest. 92, 903 – 910. Tomboc, M., Lee, P.A., Mitwally, M.F., et al., 2000. Insulin-like 3/relaxin-like factor gene mutations are associated with cryptorchidism. J. Clin. Endocrinol. Metab. 85, 4013 – 4018. Tut, T.G., Ghadessey, F.J., Trifiro, M.A., Pinsky, L., Young, E.L., 1997. Long polyglutamine tracts in the androgen receptor are associated with reduced trans-activation, impaired sperm production and male infertility. J. Clin. Endocrinol. Metab. 82, 3777 – 3782. Voutilainen, R., 1992. Differentiation of the fetal gonad. Horm. Res. 38, 66 – 71. White, R., Parker, M.G., 1998. Molecular mechanisms of steroid hormone action. Endocr. Relat. Cancer 5, 1 – 14. Wilson, J.D., Griffin, J.D., Russell, D.W., 1993. Steroid 5a-reductase 2 deficiency. Endocr. Rev. 14, 577 – 593. Wylie, C., 2000. Germ cells. Curr. Opin. Genet. Dev. 10, 410 –413. Xu, J., Qiu, Y., DeMayo, F.J., Tsai, S.Y., Tsai, M.-J., O’Malley, B.W., 1998. Partial hormone resistance in mice with disruption of the steroid receptor coactivator-1 (SRC-1) gene. Science 279, 1922 – 1925. Yeh, S., Chang, C., 1996. Cloning and characterisation of a specific co-activator, ARA 70, for the androgen receptor in human prostate cells. Proc. Natl. Acad. Sci. 93, 5517 – 5521. Young, C.Y.F., Montgomery, B.T., Andrews, P.E., Qui, S., Bilhartz, D.K., Tindall, D.J., 1991. Hormonal regulation of prostate-specific antigen messenger RNA in human prostate adenocarcinoma cell line LNCaP. Cancer Res. 51, 3748 – 3752. Zhan, Z.-X., Sar, M., Simental, J.A., Lane, M.V., Wilson, E.M., 1994. A ligand-dependent bipartite nuclear targeting signal in the human androgen receptor. J. Biol. Chem. 269, 13 115 – 13 123. Zimmermann, S., Steding, G., Emmen, J.M., et al., 1999. Targeted disruption of the Insl3 gene causes bilateral cryptorchidism. Mol. Endocrinol. 13, 651 – 691.
Developmental aspects of androgen action I.A. Hughes a,*, H.N. Lim a, H. Martin a, N.P. Mongan a, L. Dovey a, S.F. Ahmed b, J.R. Hawkins c a
Department of Paediatrics, Uni6ersity of Cambridge, Addenbrooke’s Hospital, Box 116, Cambridge CB2 2QQ, UK b Department of Child Health, Royal Hospital for Sick Children, Glasgow G3 8SJ, UK c Incyte Genomics, Cambridge Science Park, Cambridge CB4 0WA, UK
Abstract The formation of a testis from the indifferent gonad is the prelude to sequential steps in male sex differentiation orchestrated by time-dependent androgen biosynthesis and action. Information about the cellular and molecular mechanisms of androgen action can be obtained by the study of disorders of sex differentiation in males. The pivotal role of the androgen receptor as a ligand-induced transcription factor is emphasised and preliminary studies are described which attempt to identify developmentally regulated androgen-responsive genes. That androgen action can be modulated by gene polymorphisms is illustrated by the influence of an androgen receptor polyglutamine repeat in the multi-factorial causation of less severe forms of male undermasculinisation. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Sex differentiation; Androgen receptor; Co-regulators; Androgen-responsive genes; Polyglutamine repeats
1. Introduction The determination of a testis from the bipotential, indifferent gonad is critical in the male for subsequent differentiation of the internal and external genitalia. The factors involved in testis determination have been the subject of several recent reviews (Goodfellow and Lovell-Badge, 1993; Graves, 1998; Lim et al., 1998; Wylie, 2000; Quigley, 2001). The developmental aspects of androgen action are thus the consequence of differentiated Leydig cells synthesising sufficient quantities of androgen to act on target tissues during early fetal life. Knowledge about this process can be gleaned indirectly by the study of disorders of sex differentiation associated with insensitivity to the action of androgens, the syndromes of androgen insensitivity (AIS).
2. Aspects of embryology Fig. 1 depicts the major events in male fetal developmental in a temporal fashion. The first event following * Corresponding author. Tel.: + 44-1223-336885; fax: + 44-1223336996. E-mail address: [email protected] (I.A. Hughes).
testis determination is the regression of Mullerian ducts under the influence of anti-Mullerian hormone (AMH), a member of the transforming growth factor b family and secreted by the Sertoli cells. This hormone signals through binding to an AMH Type II receptor expressed in the mesenchyme of Mullerian ducts to effect regression of these structural anlage by 10 weeks of fetal age (Josso et al., 1997; Belville et al., 1999; Allard et al., 2000). The onset of testosterone synthesis is initially gonadotrophin-independent before becoming placental hCG-dependent and thereafter under the control of fetal pituitary luteinizing hormone. Concentrations of testosterone in fetal serum increase to within the adult male range and coincide with the period of Wolffian duct stabilisation in the male (Voutilainen, 1992). Fetal testes during the second trimester show histological evidence of Leydig or interstitial cell hyperplasia and immunohistochemistry reveals the expression of steroidogenic enzymes for testosterone synthesis and the androgen receptor (AR) for androgen action (Codesal et al., 1990; Murray et al., 2000). The role of androgens in stabilising Wolffian ducts to differentiate as the internal male genital ducts (vas deferens, epididymis and seminal vesicles) is based primarily on the results of fetal castration experiments in rabbits (Jost,
0303-7207/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 3 - 7 2 0 7 ( 0 1 ) 0 0 6 2 2 - 0
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1953). Unilateral castration is associated with destabilisation of Wolffian ducts which can be corrected by unilateral local application of testosterone. The preservation of Wolffian ducts in some patients with a defect in androgen biosynthesis or in responsiveness to normal androgen concentrations suggests that trophic factors in addition to androgens may play a role in Wolffian duct stabilisation (Boehmer et al., 1999; Gupta, 1996; Hodapp and Hughes 1999). The absence of androgens, however, appears to be the sole explanation for Wolffian duct regression in the female. Androgens also play a key developmental regulatory role in the differentiation of the male external genitalia. The external genital anlage are indifferent until 8 weeks of gestation when, in the male under the influence primarily of dihydrotestosterone (DHT), the genital tubercle elongates to form the penis, the urethral folds fuse to develop the penile urethra and labioscrotal folds fuse posteriorly to form the scrotum. The evidence for DHT-dependence is based on expression of the 5a-reductase Type II enzyme in these tissues (Thigpen et al., 1993) and the failure of fetal external genital development in males who lack this enzyme (Wilson et al., 1993). Development of the prostate gland is also dependent on DHT. Continued growth of the penis in later fetal life requires testosterone, synthesised now by the testis under the control of LH secretion by the fetal pituitary. Micropenis may be the consequence of prenatal anterior pituitary deficiency. Descent of the testes into the scrotum by the end of human gestation is the final step in the regulation of male fetal development. Migration of the testis from the lower pole of the kidney into the scrotum is a two-stage process of transabdominal and inguinoscrotal descent (Hutson and Beasley, 1992). Degeneration of the cranial suspensory ligament which attaches the testis to the lower pole of the kidney is part androgendependent (Emmen et al., 1998). The gubernaculum develops from the caudal suspensory ligament and is
critical for testicular descent. Its development is not androgen dependent and in the mouse, appears to be controlled by insulin-like factor 3 (insl3). Insl3 − / − mice have intra-abdominal testes and poorly formed gubernacula (Zimmermann et al., 1999; Nef and Parada, 1999). Recent studies in boys with bilateral cryptorchidism indicate that mutations in the human INSL3 gene associated with this disorder are rare (Koskimies et al., 2000; Tomboc et al., 2000; Lim et al., 2000). The final stage of inguinoscrotal descent also appears to be androgen-dependent, based on evidence of undescended testes in association with hypogonadotrophic hypogonadism and the siting of testes in syndromes associated with androgen insensitivity (Lim et al., 2001c).
3. Mechanism of androgen action Male sex development is co-ordinated by ligand activation of the AR, a nuclear transcription factor which controls androgen-dependent gene expression. The AR is ubiquitously expressed, including tissues which differentiate as the internal and external male genitalia. Current evidence indicates that a single AR binds all androgens intracellularly in target cells (Fig. 2). A single copy gene on chromosome Xq11–12 encodes the AR. Circulating androgens dissociate from carrier proteins, specifically sex hormone binding globulin, prior to diffusing into target cells and binding to the intracellular AR. The unliganded receptor is an inactive oligomer complexed to heat shock proteins (eg Hsp90, Hsp70) and located in the cytoplasm. The oligomeric complex dissociates when bound to hormone, the receptor undergoes a conformational change while transporting to the nucleus and binding as a homodimer to DNA hormone response elements (White and Parker, 1998). Activation of gene transcription via these specific hormone response elements is also a feature of the
Fig. 1. Embryologic events in male sex differentiation depicted in temporal fashion. The line depicts the increase in fetal serum testosterone concentrations.
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Fig. 2. Schematic of androgen receptor action.
related receptors for the glucocorticoid, mineralocorticoid and progestogen classes of hormones. This subfamily of nuclear receptors is but a small component of a large super-family of nuclear receptors which include receptors for other classes of steroids (oestrogens, vitamins D), thyroid hormones retinoids, thiazolidenediones (PPARg receptor) and a large group of receptors whose ligands are not known (orphan receptors). In common with other nuclear receptors, the AR is characterised by three functional domains involved in the regulation of transcription and binding to DNA and hormones (Fig. 3). A large N-terminal domain is the least conserved amongst the nuclear receptor family and contains an activation function (AF-1) region which is autonomously involved in gene transactivation. A unique feature of the AR is a polymorphic glutamine region in the N-terminus which is the result of a variable number of CAG repeats toward the 5% end of the AR gene first exon. There is evidence that variations in the length of the polyglutamine tract have effects on AR transcriptional efficiency (see later). The central DNA-binding domain is the most conserved region; the C-terminal part of the receptor contains the ligand-binding domain within which is a second activation function region (AF-2), also involved in gene transactivation (Jenster et al., 1991). Other functions mediated by the C-terminus include heat shock protein interactions, dimerisation, nuclear localisation signalling as well as ligand binding. The activation function (AF) regions interact with an intermediary group of proteins in order that transcriptional regulation of specific genes via the general transcriptional machinery be initiated (Raeder, 1996). This group of proteins, termed co-regulators, form protein:protein interactions in a ligand-dependent manner to either increase (co-activator) or decrease (co-repressor) gene transcription (McKenna et al., 1999; Robyr et al., 2000). Fig. 4 illustrates the interaction of
ligand-bound AR homodimers in a multiprotein complex with SRC-1, a representative member of the nuclear receptor coregulator family (Onate et al., 1995). The function of AF-2 is ligand-dependent and is located within one of the alpha-helices (helix 12) which binds to receptor-interacting motifs (LXXLL; L is leucine, X is any amino acid) of co-regulators (Darimont et al., 1998). This model appears to be generic to the mechanism of action of all ligand-activated nuclear receptors. In addition, the AR is unique in displaying constitutive activity in vitro when experiments involving deletion of the ligand-binding domain were performed (Jenster et al., 1991; Zhan et al., 1994). This suggests a critical role for the ligand-independent AF-1 region in gene transactivation in the case of the AR. In this instance, the interaction with the SRC-1 co-regulator appears not to be via LXXLL motifs but through interaction with a conserved, glutamine-rich region in the C-terminal region of SRC-1 (Bevan et al., 1999). Also depicted in Fig. 4 as part of the multiprotein complex is SRA (steroid receptor RNA activator) which is selective for the AF1 region of steroid receptors and uniquely functions as an endogenous RNA transcript (Lanz et al., 1999). Another co-regulator which appears to be relatively specific for interaction with the ligand-binding domain of the AR is ARA70 (Yeh and Chang, 1996; Gao et al., 1999).
Fig. 3. Structure – function characteristics of the androgen receptor. The letters denote the eight exons of the AR gene and the arrows delineate the three main functional domains of the AR. Glutamine (Gln) and glycine (Gly) are polymorphic regions in the N-terminus. AF-1, activation function 1; AF-2, activation function 2.
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Fig. 4. Schematic of ligand-bound AR interacting with co-regulator proteins. p160/SRC-1 (steroid receptor coactivator 1), CBP (CREBbinding protein), pCAF (CBP-associated factor) are representative examples of the large superfamily of coregulators. SRA (steroid receptor RNA activator) functions as an RNA transcript. DHT, dihydrotestosterone.
Androgen action in target cells is mediated via a complex pathway comprising multiple steps and interactions involving ligand, protein:protein and protein:DNA binding. Information about the critical steps in this pathway can be gained by studying XY sex reversed patients whose phenotype is the result of insensitivity to action of normal or increased concentrations of circulating androgens.
4. Syndrome of androgen insensitivity The androgen insensitivity syndromes (AIS) are defined by the complete (CAIS) or partial (PAIS) absence of signs of androgen responsiveness in genetic (XY) males with normal testis determination and androgen biosynthesis (Quigley et al., 1995; Ahmed et al., 2000a). AIS is the clinical paradigm of a hormone resistance syndrome of which there are now numerous examples relating to both nuclear receptor and cell membrane receptor-related cell signalling systems (Jameson, 1999). Characteristic features of CAIS include complete XY sex reversal in the form of a normal external female phenotype apart from the absence of, or scanty growth of, pubic and axillary hair. Internal female genital ducts are absent as the result of the action of AMH secreted by the testes; there is a blindending, shortened vagina whose development is stabilised independent of AMH or oestrogen effects. There may be variable descent of the testes but in CAIS, they are typically located in the labia. When some degree of masculinisation has occurred, such as clitoromegaly, this defines the partial form of AIS (PAIS). The external phenotype in PAIS can be variable but a common manifestation is micropenis, severe perineoscrotal hypospadias and a bifid scrotum which may or may not contain testes. A number of classification systems exist which attempt to quantify the degree of under-masculinisation (Quigley et al., 1995; Cameron et al., 1997; Ahmed et al., 2000b). A
Fig. 5. Cambridge Intersex Database. Diagnostic categories represented in the sample collection.
form of PAIS is also recognised where male factor infertility is the sole manifestation in an otherwise normally masculinised male (Giwercman et al., 2001). Numerous mutations distributed throughout the AR gene have now been reported and they are detailed on an international database (Gottlieb et al., 1998http// www.mcgill.ca/androgendb/). The Cambridge Intersex database was established to gather clinical, biochemical, histological and molecular information on XY intersex cases. As a result of a successful collaboration with numerous clinicians in the UK and mainland Europe, data is now available on more than 1000 cases. Fig. 5 illustrates the diagnostic categories, defined primarily on clinical criteria. There is a bias towards AIS because of the research interests of the Department, but the database has a representative number for other causes of XY sex reversal such as gonadal dysgenesis syndromes and defects in androgen biosynthesis. Mutations in the AR gene can be identified in the majority of patients with clinical and biochemical evidence of CAIS. In contrast, only a minority of patients with PAIS as defined by under-masculinisation in the absence of any abnormality in androgen synthesis, have mutations identified in the AR gene. A representative sample of AR mutations from the Cambridge Intersex database is shown in Fig. 6. The mutations are distributed throughout the gene and comprise a mix of missense, nonsense, deletions and insertions. There is a preponderance of mutations which affect the ligand
Fig. 6. Mutation analysis of the AR gene in AIS from samples in the Cambridge Intersex Database. Mutations are located according to exons A-H and type indicated by the symbols.
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binding domain of the AR. Functional analysis of these mutations provides indirect evidence about critical regions of the AR in support of the recently reported crystal structure of the AR ligand binding domain (Matias et al., 2000). The crystal structures of several other nuclear receptor ligand binding domains have also been determined but not yet for a complete receptor (Moras and Gronemeyer, 1998; White and Parker, 1998). They have in common 12a – helices arranged in anti-parallel fashion in three layers in the form of a sandwich fold. Helix 12 is the most C-terminal and in the presence of ligand becomes realigned to form a hydrophobic cleft where the LXXLL motifs of certain co-regulators bind. In the case of the AR, the critical regions for ligand binding can be discerned by studying the functional consequences in vivo and in vitro of missense mutations affecting the ligand binding domain. For example, using homology modelling based on the known crystal structure of the progesterone receptor, arginine 779 is critical to ligand binding and subsequent transactivation whereas a histidine 874 alanine substitution has only a minimal effect on androgen binding (Poujol et al., 2000). Mutations in the AR ligand binding domain appear to cluster in halices H4 and H5 which is the region involved in ligand binding.
5. Beyond the androgen receptor CAIS is a clearly defined disorder based on clinical features, biochemical evidence of LH-induced androgen hypersecretion and Leydig cell hyperplasia. Typically, there is absent specific AR binding in genital skin fibroblasts and a deleterious mutation of the AR gene is usually identified. The positive yield in identifying mutations in the AR gene in the PAIS form is much less, even when all the evidence points to androgen resistance causing the phenotype. This raises the possibility that defects extraneous to the AR may also manifest as androgen resistance. Two areas worthy of exploration are abnormalities in the family of nuclear co-regulator proteins and secondly, a defect in a gene responsive to androgen-induced AR transcriptional activation.
5.1. Co-regulator dysfunction Compelling evidence for the role of co-regulators mediating the optimal effect of ligand-activated nuclear receptors came from the results of disrupting the SRC-1 gene in mice (Xu et al., 1998). Target organs which are sex hormone dependent such as the mammary gland, uterus, prostate and testis showed reduced growth responsiveness in vivo to sex steroid administration compared with mice containing an intact SRC-1 gene. Also observed was over-expression of a related co-regulator, TIF2 (also known as SRC-2), perhaps in part compen-
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sation for the loss of SRC-1 activity. Further studies in the null mutant mice showed evidence also for thyroid hormone resistance based on elevated serum TSH concentrations in the face of serum thyroxine concentrations higher than in the serum of wild-type mice (Weiss et al., 1999). Hypothyroidism was induced in normal and mutant mice using a diet low in iodine and containing propylthiouracid. Serum TSH concentrations were elevated to a similar degree but the negative feedback regulation of TSH by exogenous thyroxine administration was much less sensitive in the SRC-1 knock-out mice. These animal studies by analogy suggest that some human nuclear hormone receptor resistant states may be caused by co-factor dysfunction in cases where the cognate receptor is intact. Only a few studies of co-regulators in human hormone resistance syndromes are reported to date. The report of two sisters with clinical and biochemical resistance to glucocorticoids, mineralocorticoids, and androgens but not thyroid hormones was postulated to be caused by a co-activator defect. However, no analyses were performed on any known co-activator (New et al., 1999). More specifically, a recent case report described a patient with classical features of CAIS in whom the AR gene was normal (Adachi et al., 2000). In a series of experiments using truncated and chimeric nuclear receptor constructs to assess the functional interactions between the receptors in genital skin fibroblasts and several co-activator proteins, the transmission of a transactivation signal from the AF-1 region was impaired in fibroblasts from the patient. An unknown protein corresponding to a 90 kD band which interacted with the AF-1 region of the AR was present only in fibroblasts from controls. Clearly, while further studies are required to determine the nature of this protein which appears to be a specific co-activator for the AR, the observation does raise the possibility of a novel explanation for some forms of androgen resistance (Hughes, 2000). One co-activator shown to be relatively AR-specific in human prostate cells is ARA70 which is highly homologous to the thyroid expressed RET-fused gene and displays ligand- dependent interaction with the AR (Yeh and Chang, 1996). In co-transfection assays, ARA70 acts as a relatively weak coactivator (Gao et al., 1999). We postulated that any dysfunction of ARA70 in humans could result in a milder degree of undermasculinisation compared with the phenotype produced by a deleterious AR gene mutation. The ARA70 cDNA was screened by single stranded conformational polymorphisms and heteroduplex analysis in a group of XY patients with varying degrees undermasculinisation in whom defects in the AR had been excluded (Lim et al., 2001a). No mutations which changed the amino acid sequence of ARA70 were identified or any other type of mutation. The large family of
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Fig. 7. Suppression subtractive hybridisation: differential screening of clones to determine preliminary expression profiles in cDNA populations from genital skin fibroblasts: (a) transcript in untreated cDNA population (a putative androgen repressed sequence; (b) transcript in treated cDNA population (a putative androgen induced sequence); (c) transcript in both cDNA populations, but at differing levels of transcription.
nuclear co-regulator proteins play a significant role in transcriptional regulation in a combinatorial and ligand-dependent manner. Whether the action of any one member is so specific as to cause a hormone resistance state when dysfunctional has yet to be determined in humans.
5.2. Androgen-responsi6e genes Little is known about target genes which respond to androgens, either developmentally or postnatally at the time of puberty. Yet there is an abundance of biological actions of androgens which must depend on the up-regulation of target specific genes and protein expression. Some are well characterised such as the prostate-specific proteins, probasin and prostate specific antigen (Rennie et al., 1993; Young et al., 1991). We have utilised the expression-based cloning method, Suppression Subtractive Hybridisation (SSH), (Diatchenko et al., 1996) to generate cDNA libraries enriched for androgen-regulated transcripts. To perform SSH, two cDNA populations are synthesised and PCR strategies utilised to selectively amplify sequences that are unique to either population. The cDNA populations were synthesised from genital skin fibroblasts which express high levels of the AR. Cells were cultured under carefully defined growth conditions, to limit the introduction of androgens from standard culture medium and separately treated with dihydrotestosterone and the synthetic androgen, mibolerone. After
SSH, the resultant cDNA libraries are normalised and enriched for androgen-induced and repressed sequences from the treated and untreated cultures, respectively. To determine the expression profile of individual sequences within the libraries, all sequences were cloned in bacterial hosts and arrayed for radioactive hybridisation with cDNA from treated and untreated cultures, both pre- and post-subtraction (Fig. 7). This allowed false positive clones to be removed and preliminary expression profiles to be determined. The expression profile of individual candidate clones was confirmed by Southern and Northern hybridisation prior to sequencing and further analysis (Fig. 8). Using these techniques, we have isolated a number of transcripts that demonstrate differential expression profiles between androgen treated and untreated fibroblast cultures (Fig. 8, Clone 1). In addition to sequences that are unique to either the treated or untreated cDNA populations, we have also isolated sequences present in both populations but at differing concentrations (Fig. 8, Clone 2). This suggests that dose sensitive transcription effects may also feature in androgen regulation of gene activity. The use of SSH and additional expression-based cloning techniques allows a catalogue of differentially and quantitatively expressed sequences that demonstrate androgen regulation to be compiled. The specific developmental pathways regulated by androgens may be better characterised on identification and isolation of such expressed sequences.
I.A. Hughes et al. / Molecular and Cellular Endocrinology 185 (2001) 33–41
Fig. 8. Southern hybridisation of two candidate clones to cDNA target DNA synthesised from androgen treated and untreated fibroblasts. Arrow indicates differentially expressed sequence in Clone 1 hybridisation. Clone 2 demonstrates quantitative differences in expression.
6. Factors which modulate androgen action The importance of optimal androgen production and action during the critical developmental phase of male sex differentiation has been emphasised. There is increasing evidence that polymorphic variants of gene products may modulate hormone function. In the case of androgens, a variable number of glutamine repeats in the N-terminal region of the AR is associated with disorders affecting the male reproductive system. Thus a hyper-expanded AR polyglutamine tract which leads to the neurodegenerative disorder, spinal and bulbar muscular atrophy, also results in androgen dysfunction manifest as mild androgen insensitivity, testicular atrophy and decreased spermatogenesis (La Spada et al., 1991). However, variations in the number of glutamines within the normal range (11– 31 repeats) are associated with male reproductive disorders such as decreased
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spermatogenesis in otherwise normal males (Tut et al., 1997; Dowsing et al., 1999). In vitro studies indicate that an AR containing a longer polyglutamine tract is transcriptionally less active; indeed, the opposite in vitro observation is the case with shorter tracts. There is conflicting evidence that a shorter AR glutamine repeat may be not only a risk factor for developing prostate cancer but also influence the age of onset and response to treatment involving endocrine ablation (Stanford et al., 1997; Bratt et al., 1999; Correa-Cerro et al., 1999). We have previously demonstrated that longer polyglutamine repeats within the normal range are associated with varying degrees of undermasculiniation of unknown cause (Lim et al., 2000) (Fig. 9). The estimated increase in the odds ratio for each additional repeat was 9.1%. In a subsequent study of a larger number of males with abnormal genital development, we found evidence that a longer AR glutamine repeat may contribute to the cause of genital maldevelopment, particularly with less severe genital abnormalities (Lim et al., 2001b). Furthermore, there was a strong association between decreasing severity of the genital malformations and an increasing proportion with a multifactorial aetiology. On the basis of these findings, a model for the involvement of the AR polyglutamine length in genital abnormalities is proposed (Fig. 10). It suggests that the relative impairment of AR transcriptional activity consequent upon a longer repeat is a contributory cause in a multifactorial fashion to the syndrome of moderate undermasculinisation. In contrast, when hormone resistance is absolute (complete sex reversal as in CAIS), the contribution of the longer AR glutamine repeat to the genital malformation is negligible. Further studies of larger patient populations analysing multiple genes involved in the control of androgen production and action will contribute to a better understanding of the developmental aspects of androgen action in the human.
Fig. 9. Distribution of AR glutamine repeats in undermasculinised subjects compared with controls.
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I.A. Hughes et al. / Molecular and Cellular Endocrinology 185 (2001) 33–41
Fig. 10. A model incorporating the effect of an AR polymorphism on the aetiology of genital abnormalities. The influence of a longer glutamine repeat is greater when multi-factorial causes lead to moderate genital abnormalities.
Acknowledgements The support of the Birth Defects Foundation, European Community, Sir Halley Stewart Trust and the Cambridge Children’s Kidney Care Fund for some of the studies described in this paper is gratefully acknowledged.
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