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725 BIOLOGY OF REPRODUCTION 59, 725–732 (1998) Developmental Expression of the Androgen Receptor during Virilization of theUrogenital System of a Marsupial 1 Christopher M. Butler, 2,4 Jenny L. Harry, 3,4 Janine E. Deakin, 5 Desmond W. Cooper, 5 and Marilyn B. Renfree 4 Department of Zoology, 4 University of Melbourne, Parkville, Victoria 3052, AustraliaThe Marsupial Collaborative Research Centre, 5 School of Biological Sciences, Macquarie University, North Ryde,NSW 2019, Australia ABSTRACT In the marsupial tammar wallaby, virilization begins approx-imately 3 wk after the onset of testosterone synthesis. In theeutherian mammal, in contrast, the onset of virilization of themale urogenital tract occurs shortly after the onset of androgensynthesis. Androgen action requires the presence of the andro-gen receptor to mediate a response in target tissues. We there-fore investigated the developmental expression of the androgenreceptor (AR) in both sexes of the tammar wallaby. AR genetranscript was detected in fetal gonad and brain as early as Day19 of the 26.5-day gestation, 7 days earlier than the first rise intesticular testosterone (Days 0–5 postpartum [p.p.]). Immuno-reactive AR was identified in the male urogenital sinus (UGS) 2days before birth and in the female UGS and mammary glandsby the day of birth. AR was present in the UGS, vagina, andprostate until Day 152 p.p., the oldest age examined. AR wasidentified in the gubernaculum testis at Day 2 p.p. and becamemore abundant by Day 32. In the phallus of both sexes, AR wasidentified by Day 4 p.p. and until Day 157, the oldest age ex-amined. AR was not detected in the scrotum at any age fromthe day of birth to Day 157. Maturation of the phallus, wolffianduct, and epididymis was marked by appearance of epithelialimmunostaining. AR was localized in the epithelium of the UGSin females by Day 50 p.p. but was not found in the epitheliumof the male UGS up to Day 152 p.p., the oldest examined. ARwere found in the mesenchyme of the UGS of male and femaletammars 3–4 wk before virilization is first evident in the maleat Day 25 p.p. We conclude that the presence of AR is not theinitiating signal for virilization of the UGS in this marsupialmale. INTRODUCTION In mammals, the morphological events leading to thevirilization of the male reproductive tract are controlled bythe androgenic hormones testosterone (T) and dihydrotes-tosterone (DHT) [1]. T controls differentiation and growthof the wolffian duct derivatives (vas deferens, epididymi-des, and seminal vesicle), while its 5 -reduced metaboliteDHT is responsible for prostate development and viriliza-tion of the external genitalia [2, 3].T and DHT both act within cells via interaction with thesame androgen receptor (AR) [2]. The AR is a member of Accepted April 23, 1998.Received November 20, 1997. 1 This study was supported by grants from the National Health andMedical Research Council of Australia to M.B.R., R.V. Short, and G. Shaw,and from the Australian Research Council and the New Zealand Ministryof Agriculture to D.W.C. 2 Correspondence. FAX: 61 3 93447909;e-mail:
[email protected] 3 Current address: Australian Proteome Analysis Facility, School of Bi-ological Sciences, Macquarie University, North Ryde, NSW, 2019, Aus-tralia. the superfamily of ligand-binding transcription factors thatincludes receptors for steroid hormones, thyroid hormone,vitamin D, and retinoic acid [4]. DHT has a higher bindingaffinity and slower rate of dissociation from the AR thandoes T [5]. Thus, the androgenic effect is amplified by theconversion of T to DHT. The control of cellular differen-tiation by androgens is therefore dependent on the presenceat target sites of hormone, AR, and, in some tissues, 5 -reductase.In the marsupial tammar wallaby ( Macropus eugenii ),the scrotum, mammary primordia, gubernaculum, and pro-cessus vaginalis differentiate during early fetal life underthe influence of a gene or genes on the X chromosome,well before the testis becomes functional [6–9]. The re-maining sexually dimorphic structures, namely the vasa de-ferentia, epididymides, prostate, male urethra, and phallus,are all under androgenic control and differentiate postna-tally after the formation of testis cords at Day 2 postpartum(p.p.) and the onset of testicular T production [10]. Thedevelopment of the prostate can be prevented by treatmentwith the androgen receptor inhibitor, flutamide, or with fi-nasteride, which inhibits DHT formation, during the periodof virilization of the urogenital sinus [11, 12]. Prostate for-mation in females is stimulated by exogenous T [9, 12].Castration of early pouch young inhibits the formation of the penis and prostate in males, and transplantation of testesto female pouch young induces formation of the penis andprostate [13].Pre-Sertoli cells can be identified at the ultrastructurallevel 1 day before birth in the tammar. Testis cords are firstseen at a light microscopic level 2 days after birth coincid-ing with measurable gonadal T [14]. Testicular T level risesto plateau at around Day 10 and remains high until Day40, after which it gradually declines to basal levels by Day70 [10]. T remains relatively low until puberty, which oc-curs between 19 and 25 mo of age in this species [15]. 5 -Reductase activity, as measured by DHT production in cul-tured tissues, is detectable at a high level in the urogenitalsinus (UGS) and urogenital tubercle from at least Day 10p.p. [10].Virilization of the UGS of the tammar is androgen de-pendent and requires the presence of both DHT and activeAR [11, 12]. Sensitivity to androgens is developed in theUGS in a relatively narrow window of time between Days20 and 25 p.p. when the prostatic buds first appear [9, 11,12, 16]. These epithelial buds, the first sign of virilizationof the UGS, are not evident until 3 wk after initiation of Tproduction, which occurs immediately after birth. In con-trast, in eutherian mammals, virilization of the UGS andwolffian duct commences shortly after the initiation of Tsynthesis by the fetal testis [1]. In the rat, T productioncommences around gestational Day 15.5 and peaks at 18.5 726 BUTLER ET AL. FIG. 1. a ) Partial sequence of tammar AR gene spanning the intron/exon junction of exons 4 and 5 with putative amino acid sequence. Primersequences are underlined. b ) Putative amino acid sequence compared tothe brushtail possum ( Trichosurus vulpecula ), human, and rat sequencefor the same region. Differences are shaded. Numbering of rat and humansequence from Lubahn et al. [35]. days [17, 18]. Prostatic buds in the rat embryo first developin the UGS around Day 19.5 and continue to mature post-natally [19]. Sexual dimorphism of the wolffian duct is ev-ident by gestational Day 16 [20]. Similarly, virilization oc-curs within a few days of T production by the fetal testisin the rabbit [21–23], sheep [24], guinea pig [25], and hu-man [26]. In the rabbit, functional studies have shown thatthe fetal AR is active in the UGS from Day 18 of gestation,the time at which T synthesis begins in the fetal testis [27,28].The extensive delay between T and DHT production andvirilization in the male tammar suggests that some otherfactor is responsible for initiation of virilization. We hy-pothesized that the sensitivity to androgens in the UGScould be controlled by the presence/absence of AR, whichwould be rate-limiting for the onset of virilization. Wetherefore investigated the ontogeny of the AR in the de-veloping male tammar throughout the period of virilization.Since administration of superphysiological levels of T tofemale pouch young induces prostatic bud formation at thesame rate as in control males of the same age [9, 12], wealso assessed AR expression in the developing female. MATERIALS AND METHODS Animals Tammar wallabies of Kangaroo Island, South Australia,srcin were kept in open grassy yards and provided withlucerne cubes, oats, fresh vegetables, and water ad libitum.Tammar wallabies are seasonal breeders and have a post-partum estrus that results in the presence of a diapausingblastocyst in the uterus while a pouch young is suckled.Removal of pouch young (RPY) causes reactivation of thequiescent blastocyst. Births occur on average 26.5 days lat-er [29]. The aging of fetuses according to days after RPYinstead of days of gestation reflects active gestation afterthe blastocyst stage, since in this species total gestationnormally includes 11 mo of embryonic diapause. Adult fe-male tammars were checked daily for births. In cases inwhich the day of birth was uncertain, the new pouch youngwere aged by head length [30].Pouch young aged between 5 and 60 days were killedby decapitation; the gonads, phallus, and UGS were re-moved, embedded in Tissue-Tek OCT (Miles Laboratories,Melbourne, Victoria, Australia), and snap frozen in a dryice/ethanol slurry. Fetuses and pouch young less than 5days old were bisected at the level of the metanephric kid-ney, and the caudal half of the animal was embedded forimmunohistochemical analysis. Adult animals and pouchyoung older than 60 days were killed by overdose of so-dium pentobarbitone in sterile saline, and tissues were pro-cessed as above. Tissues for reverse transcription-polymer-ase chain reaction (RT-PCR) were snap frozen in liquidnitrogen. All samples were stored at 80 C until processed.Tissue used in RT-PCR was obtained from brains of onemale fetus at Day 22, of one fetus of each sex at Days 23,24, 25, 26 RPY, and of one pouch young of each sex at theday of birth, Day 2, and Day 4 p.p.; and from the posteriorhalf of two fetuses of each sex (Days 19, 20, 22 RPY),gonad and mesonephros (Days 23, 24 RPY), gonad only(Days 25, 26, RPY), and gonads from two pouch young of each sex at the day of birth, Day 2, and Day 4 p.p. UGS,developing gonads and associated ducts, scrotum, and phal-lus of fetuses from Day 25 RPY and pouch young fromday of birth to Day 157 of pouch life were examined forthe presence of AR protein. In all immunohistochemistryexperiments, at least two males and two females of com-parable age were used to represent each age except femaleDay 25 RPY fetus (n 1). All experiments followed theNational Health and Medical Research Council (1990)guidelines and were approved by Institutional AnimalEthics Committees. Partial Cloning and Sequencing of Tammar AR Primers for PCR (forward primer 5 CACATTGA-AGGCTATGAGTG 3 and reverse primer 5 CCCA-TCCAGGAGTACTGAAT 3 ; see Fig. 1) were based on asequence spanning exons 4 and 5 of the AR from the brush-tailed possum ( Trichosurus vulpecula ) (GenBank accessionnumber AF033557). This region is highly conserved acrossmany species. Total RNA from tammar adult prostate andepididymis (1 g) was reverse transcribed and the cDNAwas used for PCR (see below). The PCR products wereseparated on a 2% agarose gel, purified (Wizard minipreps;Promega, Annadale, NSW, Australia), and cloned using thepGEMT vector system according to the manufacturer’s in-structions (Promega). The vector was digested with Apa Iand Sac I, and the insert was sequenced using theAmpliCycle-PCR sequencing kit (Perkin Elmer, Sydney,NSW, Australia) according to the manufacturer’s instruc-tions. The PCR product from epididymis was sequenceddirectly after purification from the agarose gel. RT-PCR Total RNA was extracted from samples using the guan-idinium thiocyanate method [31] and reverse transcribedusing Mo-MuLV reverse transcriptase (Gibco-BRL, Mel-bourne, Victoria, Australia). Primers used in PCR are de-scribed above. Cycle conditions for amplification using ARprimers were 94 C, 30 sec (94 C, 30 sec; 52 C, 1 min;72 C, 1 min for 36 cycles) and 72 C, 2 min. Efficiency of the reverse transcription and PCRs was monitored by am-plification of phosphoglycerate kinase (PGK) as describedpreviously [32]. Tammar genomic DNA was used in all 727 ANDROGEN RECEPTOR LOCALIZATION IN THE WALLABY FIG. 2. Expression of AR gene transcript in developing tammar tissues.Transcribed AR mRNA produces a band at 270 base pairs (bp) andgenomic DNA at 870 bp. a ) Posterior fetus and developing gonad. Lane1, markers (bp); lanes 2–7, posterior half of fetus; lanes 8–11, gonad/ mesonephros complex; lanes 12–21, gonad only (age and sex as marked);lane 22, genomic DNA. b ) Brain. Lane 1, markers (bp); lanes 2–16, fetaland pouch young brain samples, age and sex as marked; lane 17, ge-nomic DNA; lane 18, pGEMT plasmid containing insert used to generatethe sequence in Fig. 1. c ) Representative PGK control lanes as for b . dOB,day of birth; m, male; f, female. PCR experiments to identify bands generated by contami-nation with genomic DNA. Controls lacking template DNAor Taq polymerase were also run for each sample. Immunohistochemistry Three antibodies were used to immunolocalize AR intammar tissue. Antibodies U402 (a kind gift of ProfessorJ.D. Wilson, Dr. C.M. Wilson, and Dr. M.J. McPhaul; De-partment of Internal Medicine, University of Texas, SouthWestern Medical Center, Dallas, TX) and PG21 (SignetLaboratories, Adelaide, South Australia) both recognizepart of the transactivation region at the amino terminus of human AR [33, 34]. R489 (also a gift from Professor J.Wilson, Dr. C.M. Wilson, and Dr. M.J. McPhaul) recogniz-es part of the ligand-binding region at the carboxyl terminusof the human AR [33]. These antibodies cross-react withAR in a number of other mammalian species.Tissue sections (10 m) were cut on a Reichert-Jung3CM3000 cryostat (Leica, Hawthorn East, Victoria, Aus-tralia), thaw mounted on Histogrip (Zymed Laboratories,Gymea, NSW, Australia)-coated microscope slides, andfixed for 5 min in 10% formalin in PBS, pH 7.4. Sectionswere washed briefly, postfixed in ice-cold methanol with0.3% H 2 O 2 for 5 min (to block endogenous peroxidases)and ice-cold acetone for 1 min, and washed in PBS (0.01M, pH 7.4). The immunohistochemistry method of Hus-mann et al. [33] was followed using the Vectastain ABCkit (Vecta Laboratories, Camperdown, NSW, Australia) anddiaminobenzidine (DAB) as chromogen for reaction withthe U402 antibody, and a slightly modified protocol forPG21 and R489. All antibodies were diluted in blockingserum (1% wallaby serum, 3% normal goat serum in PBS,pH 7.4) and incubated overnight at 4 C before use. U402antibody was used at a dilution of 1:400, and sections wereincubated overnight at 4 C. Secondary antibody was usedat 1:400 in blocking serum, and ABC complex was used at1:400 in PBS.The PG21 and R489 antibodies were used at 1:50 and1:400 dilutions, respectively, and sections were incubated48 h at 4 C. Sections were then washed in PBS and incu-bated with second antibody (1:400, 30 min). All washesafter use of the secondary antibody were in Tris-bufferedsaline (TBS), pH 7.5, with 0.5% Tween 20 (ICN, Mel-bourne, Victoria, Australia). Sections were then incubatedwith ABC complex (1:400 in PBS, 30 min) and washed,and the signal was amplified by incubating sections withbiotinylated tyramide (1:200 in amplification diluent; NewEngland Nuclear [NEN], Sydney, New South Wales) or 0.1M borate buffer, (pH 8.0 with 0.03% H 2 O 2 ) for 10 minfollowed by streptavidin-horseradish peroxidase (1:4000[TBS], pH 7.5, and 0.5% blocking compound, NEN) for 30min. The sections were developed using DAB as used forU402. The amplification step allowed the primary antibodyto be used at a lower concentration and improved stainingintensity without increasing background.All experiments included positive (adult prostate) andnegative (spleen) control tissues. Alternate sections of eachtissue were taken. One was treated with primary antibody,and in the other, either the primary antibody was omittedor antibody preabsorbed to the appropriate peptide wasused as a control. Preabsorption of the antibody resulted incomplete abolition of immunostaining in all cases. RESULTS AR Gene Expression The specificity of primers used in RT-PCR was estab-lished by comparing the sequence of the single bands pro-duced in PCR, using total RNA from the prostate and ep-ididymis of the adult tammar, to the known sequences of brushtail possum, human, and rat AR [35] (Fig. 1). Thetammar partial sequence showed high homology to thebrushtail possum, human, and rat sequences in this highlyconserved region of the AR (Fig. 1b). The sequences gen-erated from tammar epididymis and prostate were identical. AR gene transcript was detected in tammar fetuses asearly as Day 19 RPY, the earliest stage investigated (Fig.2a). The genital ridge arises at around Day 20 RPY, andthe gonad/mesonephros complex can be dissected by Day23 RPY. AR transcript was detected in gonad/mesonephroson Day 23 and Day 24 RPY and in gonad from Day 25RPY to Day 4 p.p., the oldest age examined (Fig. 2a). AR transcript was present in both sexes at all ages examined. AR transcript was also present in developing tammar brainfrom Day 23 RPY to Day 4 p.p. (oldest examined) in both 728 BUTLER ET AL. FIG. 3. Immunohistochemical controls. a )Adult prostate incubated with antibodyU402. All epithelial cells show positive(‘‘ginger’’) immunostaining in nuclei only(arrows). b ) Control, adult prostate incu-bated with antibody U402 preabsorbed tothe peptide used to produce the antibody. c ) Adult prostate incubated with antibodyPG21. Epithelial nuclei show immunoreac-tive AR. d ) Control, adult prostate incubat-ed with secondary antibody only. e ) Adultprostate incubated with R489 antibody;immunoreactive AR is present in both nu-cleus and cytoplasm of epithelial cells. f )Control, adult prostate incubated with sec-ondary antibody only. Bars 56 m. sexes (Fig. 2b). Control PCR reactions detecting the PGKhousekeeping gene were carried out on the same cDNAsamples as used for PCR with AR primers. In all casesexcept one (Day 22 RPY male brain), control reactionswere positive (Fig. 2c). The negative result for the Day 22RPY male brain sample using both AR and PGK primerssuggests that either the RNA extraction procedure or thereverse transcription reaction did not work. Control reac-tions using water instead of template DNA or omission of DNA polymerase were run with each reaction and werenegative in all cases (results not shown). Immunolocalization of AR Adult prostate sections (included in each experiment asa positive control tissue) showed positive immunostainingfor all three antibodies. Preabsorbing the antibody to itsrelevant antigenic peptide abolished any immunostaining inpositive controls (Fig. 3). AR immunostaining was consid-ered to be positive only if control sections (preabsorbedantibody or no primary antibody) showed no staining (Fig.3, b, d, and f). PG21 and U402 antibodies localized ARimmunostaining to the nucleus of positive cells (Fig. 3, aand c). R489 antibody staining was both nuclear and cy-toplasmic (Fig. 3e). UGS and Developing Prostate In the neonatal male, UGS AR immunostaining wasclearly present in mesenchymal tissue (Fig. 4a). There wasa weak localization in the male fetus at Day 25 of gestationapproximately 1.5 days before birth (Fig. 4b), and AR wasdemonstrable in the mesenchyme of the UGS or the stromaof the prostate (Fig. 4d) until the oldest age examined (Day152 p.p.). AR was present in both epithelium and stromaof adult prostate (Fig. 3), but in developing males the ep-ithelium of the UGS showed no or very low levels of im-munostaining of AR even after the prostate had differenti-ated. The oldest age examined was Day 152, when the glan-dular epithelium has differentiated and the ductal lumenwithin the glandular portion is beginning to expand. Epi-thelial cells remained, for the most part, AR negative exceptfor occasional cells that stained at a very low level. No ARimmunostaining was found in the posterior half of late-gestation female fetuses (results not shown). AR in the fe-male were first found in the mesenchyme of both the UGSand developing mammary glands by the day of birth (Fig.4c) and were strongly present by Day 2. Localization of AR in female pouch young was initially the same as thatin males. The older female UGS differed from that of malesin that AR immunostaining appeared in the epithelium of the UGS by approximately Day 50 of pouch life (Fig. 4e).By Day 158 when the vagina is fully differentiated, smoothmuscle within the vaginal walls and the vaginal epitheliumboth stained strongly for AR (Fig. 4f), a pattern that wasalso found in the adult vagina. Interestingly, the R489 an-tibody showed an identical tissue localization of the AR inthe early UGS, but the AR was found in the cytoplasmrather than the nucleus. Phallus AR was present in mesenchymal tissue of the phallus inpouch young of both sexes from Day 4 onward (Fig. 4g).In males, AR was found in urethral epithelium and in mes-enchyme until at least Day 61 p.p., but at Day 152 ARwere detectable in the epithelium of the urethra only (Fig.5a). In females, receptors were seen in the mesenchymaltissue and epithelium of the urethra until Day 118, the old-est age examined (Fig. 5b). Gonads and Wolffian Ducts AR was not detected in gonads of either sex except intwo males. One Day 32 male showed strong immunostain-ing in Sertoli cells and interstitial cells (Fig. 5d), and inone Day 34 male, weak immunostaining was seen in theinterstitial cells and the rete testis. In these same specimensthe wolffian ducts had differentiated into the epididymidesand vasa deferentia. Strong staining was present in the ep-ithelial lining of the vas deferens, whereas the surroundingmesenchyme was devoid of AR immunostaining. In con-trast, immunostaining once more appeared in the surround-ing outer mesenchyme (Fig. 5d, inset). At Day 157 p.p.there was strong staining in the epithelium of the epididy-mal tubules (Fig. 5e). Gubernaculum The gubernaculum, a strand of mesenchymal tissue at-tached to the base of the scrotum, was immunoreactive forAR at a low level in a 2-day-old male pouch young (Fig.5c). At Day 32 the gubernaculum was strongly AR positive(Fig. 5d). By Day 157, the remains of the gubernaculumare present as the tendon attaching the testis to the base of the scrotum, and this tissue showed a low level of AR-positive immunostaining. 729 ANDROGEN RECEPTOR LOCALIZATION IN THE WALLABY FIG. 4. a ) Male at day of birth, cross section at level of bladder. ugs, urogenital sinus; m, mesenchyme; e, epithelium. Note scrotal bulges (sc); g, gut;p, pelvis. b ) Male fetal UGS, Day 25 RPY. The UGS has formed and the bladder is developing. wd, wolffian ducts. c ) Female pouch young, Day 0–1p.p. mg, mammary gland primordia. d ) Prostate of male pouch young, Day 152 p.p. Note lumen of the glandular epithelium has not yet expanded(arrows). s, stromal cells of the prostate; vd, vas deferens. e ) Female UGS, Day 60 p.p. f ) Female lower vagina, Day 158 p.p. sm, smooth muscle. g )Female phallus, cross section, Day 4 p.p. Bars 286 m except b , bar 111 m.
