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Keywords:

  • hypospadias;
  • aetiology;
  • androgen receptor;
  • genetics;
  • signalling

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of Interest
  9. References

What's known on the subject? and What does the study add?

  • Despite diverse anatomical and histological trials in humans and animal models, the aetiology of hypospadias remains unknown and currently there is no clear molecular explanation about the emergence of this disease; however, genetic, endocrine and environmental mechanisms have been suggested.
  • The aim of the present study was to quantify and compare the androgen receptor (AR; mRNA and protein) levels in 40 prepuces of boys with and without hypospadias using quantitative real-time polymerase chain reaction, Western Blot and standardised, automated immunohistochemistry. AR mRNA (P = 0.013) and AR protein (P = 0.014) was significantly elevated in the prepuces of boys with hypospadias compared with boys without hypospadias. Altogether our data indicate that elevated AR mRNA and protein levels can be considered as a biochemical response of an AR signalling defect as an identified cause in boys with hypospadias. Additionally, nuclear staining intensity for AR-protein in specimens of boys with hypospadias was higher than in boys with phimosis.

Objective

  • To address the role of the androgen receptor (AR) on mRNA and protein levels in prepuces of boys with and without hypospadias.

Patients and Methods

  • Data from 40 foreskin specimens of consecutive circumcised boys (20 with vs 20 without hypospadias) were enrolled in this prospective study. After surgery, samples were fixed in formaldehyde and frozen in liquid nitrogen. Total RNA was isolated from frozen tissue and transcribed to complementary DNA.
  • The amount of AR mRNA was measured by quantitative real-time polymerase chain reaction and Western Blot and standardised, automated immunohistochemistry were used to assess AR protein levels.

Results

  • The mean age at time of surgery was 61.8 and 30.9 months in boys without and with hypospadias, respectively.
  • There was penile, coronal and sine hypospadias in seven (35%), nine (45%), and four (20%) boys, respectively.
  • AR mRNA was significantly elevated in the prepuces of boys with hypospadias compared with boys without hypospadias, at a mean (sd) of 28.33 (5.39) vs 15.31 (1.85) (P = 0.013).
  • Furthermore, the amount of AR protein was higher in boys with, compared with boys without hypospadias, at a mean (sd) of 133.25 (6.17) vs 100 (4.45) (P = 0.014).

Conclusions

  • Different AR mRNA expression and protein levels seem to be an indication of an AR signalling defect as a cause in boys with hypospadias.
  • Decreased AR DNA binding and functional capability may result in a compensatory up-regulation of both AR mRNA and protein.
  • Further studies are necessary to perform structural analysis of the AR and to corroborate these preliminary findings.

Abbreviations
AR

androgen receptor

cDNA

complementary DNA

Ct

cycle threshold (value)

IHC

immunohistochemistry

rtPCR

real-time PCR

TBP

TATA-box binding protein

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of Interest
  9. References

Hypospadias is one of the most common congenital anomalies, occurring in about one of 250 newborns or one in 125 live births [1], defined as a midline fusion defect of the male urethra with additional anomalies, e.g. dorsal hooded foreskin, persistent chordae, penile curvature and penoscrotal transposition [2]. Systems for categorising hypospadias are based on the location of the external meatus distinguishing distal-anterior hypospadias (located on the glans or distal shaft of the penis), intermediate-middle (penile) and proximal-posterior (penoscrotal, scrotal, perineal) [3].

Currently there is no clear molecular explanation about the emergence of this disease; however, genetic, endocrine and environmental mechanisms have been suggested [4].

The exact molecular events required in the genitourinary tract for normal development of the external genitalia are just beginning to be elucidated [5], the aim of the present study was to quantify and compare the androgen receptor (AR) levels in prepuces of boys with and without hypospadias.

Patients and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of Interest
  9. References

The study was performed after approval from the Local Ethical Committee (study number UN3645; 277/4.6). In all, 40 prepuces of boys who underwent consecutive circumcision either because of phimosis (group 1; n = 20) or because of hypospadias repair (group 2; n = 20) were included in this prospective study. After circumcision, the prepuce as a whole was divided and sliced in separate tissue samples; either frozen in liquid nitrogen and stored at –80 °C until use or fixed in paraformaldehyde and embedded in paraffin according to standard procedures.

Quantitative Real-Time PCR (rtPCR)

For isolation of total RNAs the Promega SV total RNA isolation system (Promega Corporation, Madison, USA) was used, following the manufacturer's protocol. Complementary DNA (cDNA) synthesis was performed using Superscript III RNase H-reverse transcriptase (Invitrogen, Carlsbad, CA, USA) and random primers. Quantitative rtPCR conditions were as follows: one cycle of denaturing at 95 °C for 10 min followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. All amplifications were performed in triplicate. TATA-box binding protein (TBP) was chosen as an endogenous expression standard. Primer and probe sequences were as follows: AR (forward 5′-AGGATGCTCTACTTCGCCCC-3′; reverse 5′-CTGGCTGTACATCCGGGAC-3′; TaqMan probe 5′-Fam-TGGTTTTCAATGAGTACCGCATGCACA-Tamra-3′), TBP (forward 5′-CACGAACCACGGCACTGATT-3′; reverse 5′-TTTTCTTGCTGCCAGTCTGGAC-3′; TaqMan probe 5′-Cy5-TCTTCACTCTTGGCTCCTGTGCACA-BHQ2-3′). Each reaction was performed in a 21 μL volume containing 50 ng of cDNA and 6 μL of 2x ABI Mastermix (Applied Biosystems, Foster City, CA, USA). The primers were added to a final concentration of 900 nmol/L, and the final concentration of the probe was 150 nmol/L. PCR products were measured using the ABI Prism 7500 Fast RT-PCR System (Applied Biosystems). The cycle threshold (Ct) values of AR and TBP as assessed and used to calculate the ΔCt values and relative concentration were calculated in Microsoft Excel 2002 using the formula 2−ΔCt.

Immunoprecipitation and Western Blot

Tissue was lysed in RIPA buffer containing 1%Triton X-100, protease, and phosphatase inhibitors (Invitrogen). The total protein was quantified using the Bradford method [6]. In all, 400 μg of protein was used to precipitate AR with a rabbit anti-AR antibody (1:200, Cell Signaling Technology, Danvers, MA, USA) by using Pansorbin Cells (Calbiochem, EMD Chemicals inc., La Jolla, CA, USA) according to manufacturer's protocol. Electrophoresis was carried out using a 4–12% Bis-Tris gel (Invitrogen, Leek, The Netherlands) followed by transfer onto a nitrocellulose membrane (Invitrogen). The membrane was blocked for 1 h using StartingBlock (TBS) buffer (Pierce Biotechnology, Rockford, IL, USA) and incubated at 4 °C overnight with a mouse anti-AR antibody (1:1000, clone F39.4.1, Biogenex Laboratories, San Ramon, CA, USA). This step was followed by incubation for 1 h at room temperature with fluorescence-labelled secondary antibodies (Molecular Probes, Eugene, OR, USA). The membranes were scanned and quantified using the Odyssey infrared imaging system (LiCor Biosciences, Lincoln, NE, USA).

Immunohistochemistry (IHC)

The tissue samples were fixed in 4% paraformaldehyde in PBS (0.1 m) for 6–9 h and afterwards rinsed in the same buffer. Subsequently, the prepuces were dehydrated in graded isopropanol and xylene series and embedded in paraffin. Histological sections (4 μm) were cut on an HM 355 S microtome (Microm, Walldorf, Germany). IHC analysis for immunoreactivity of AR was performed using standardised automated procedures (Discovery XT, Ventana Medical Systems, Tucson, AZ, USA). Slides were deparaffinised; antigen retrieval was achieved by heating at 95 °C for 60 min in Cell Conditioner 1 retrieval buffer (pH 7.8, Ventana Medical Systems). Monoclonal mouse antihuman AR antibody (clone AR441, DAKO, Glostrup, Denmark) diluted 1:25 in PBS was used as primary antibody and incubated for 4 h. This step was followed by incubation with a ready to use secondary biotinylated antibody (Ventana Medical Systems) for 1 h. After washing, streptavidin-horseradish peroxidase was applied for 16 min. Finally the antigen-antibody complex was visualised using the diaminobenzidine (DAB) detection kit for 8 min. Counterstaining was performed with haematoxylin. All control incubations without application of the primary antibody yielded no signal.

IHC on human prostate tissue was performed as a positive control with quantitative analysis using TissueFAXS and HistoQuest IHC analysis software.

Statistics

Statistical analysis was performed using the Kolmogorov-Smirnov test, unpaired Student's t-test and Mann–Whitney U-test. The results are expressed as the mean (sd) and considered statistically significant when P < 0.05 and highly significant when P < 0.01.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of Interest
  9. References

The mean (sd, range) age of the boys in group 1 (phimosis) at the time of surgery was 61.8 (8.5, 18–162) months and in group 2 (hypospadias) was 30.9 (7.9, 13–156) months. In group 2 (hypospadias, n = 20), the urethral meatus was found coronal in nine (45%) and distal penile in seven (35%) boys. ‘Hypospadias sine hypospadias’ were observed in four (20%) boys and were characterised as hypospadias with a ventral curvature of the penile shaft, dorsal hood but with an orthotopic position of the meatus.

To evaluate the amount of AR expression in the analysed prepuces, we first measured its mRNA using quantitative rtPCR. AR mean (sd) mRNA levels were significantly higher in the prepuces of boys who had hypospadias (28.33 [5.39]) compared with boys undergoing surgery for phimosis (15.31 [1.85]), (Fig. 1, P = 0.013).

figure

Figure 1. AR mRNA levels were elevated in the hypospadias group compared with the phimosis group. AR mRNA levels were determined by quantitative rtPCR, TBP was used as an endogenous control. Statistical analysis showed a statistical higher expression of AR-mRNA in specimens of boys who had hypospadias (P = 0.013). Error bars represent sd of independent experiments. Mann–Whitney U-test; **P < 0.01.

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Next we investigated the AR protein levels using Western blot and IHC. AR-protein was successfully immunoprecipitated from protein extracts of prepuces tissue and visualised and quantified by Western blot analysis. A single band AR-protein corresponding to the expected molecular weight of 110 kDa was detected, scanned and quantified. The mean (sd) AR-protein level was 100 (4.45) in the phimosis group and 133.25 (6.17) in the hypospadias group. There was a significant elevation of AR protein in boys with hypospadias compared with healthy boys (Fig. 2, P = 0.014).

figure

Figure 2. AR protein levels are elevated in boys with hypospadias compared with those with phimosis. AR-protein levels were determined in prepuce tissue protein extracts by immunoprecipitation followed by Western blotting. Densitometric analysis and results of representative samples are shown (*P = 0.014). Error bars represent sd of independent experiments. Mann–Whitney U-test; **P < 0.01.

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Additionally, tissue specimens were analysed by IHC. The immunoreaction of AR-protein in BPH was included as a positive control and non-immune serum as a negative control. IHC showed that cells staining for AR-protein were predominantly found in the basal layers of the epidermis and were totally missing in the subepidermal layers. In all specimens positive staining for AR-protein was detectable, but staining intensity differed from group 1 to group 2. Nuclear staining intensity for AR-protein in specimens from boys with hypospadias (Fig. 3B) was higher than in boys with phimosis (Fig. 3A).

figure

Figure 3. IHC staining for AR in boys without (A) and with (B) hypospadias. AR levels were elevated in the prepuce specimens of boys with hypospadias compared with those with phimosis. IHC on human prostate tissue was performed with quantitative analysis using TissueFAXS and HistoQuest IHC analysis software. Scale bar 20 μm.

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In summary, both the AR mRNA and protein levels were higher in the genital skin of boys with hypospadias compared with healthy boys.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of Interest
  9. References

Despite diverse anatomical and histological trials in humans and animal models, the aetiology of hypospadias remains unknown. The present study was undertaken to investigate the role of the AR in the development of hypospadias. Familial clustering suggests a genetic cause in the incidence of hypospadias in boys. For example, a study found that the risk of a second male sibling being born with hypospadias when this anomaly occurred for the first time in the index child was 11%. Thus, a polygenetic, multifactorial mechanism of inheritance is suggested [7].

However, most patients show a sporadic malformation without an obvious underlying cause. A correlation between the severity of hypospadias malformation and the underlying reasons could not be identified [4, 8].

Additionally, different genes involved in testicular determination (Wilms' tumor 1, WT1; sex-determining region of the Y chromosome, SRY), penile development (Homeobox, HOX; fibroblast growth factor, FGF; Sonic hedgehog, Shh), and regulation of the synthesis (LH receptor) and action of androgen (5α-reductase, AR) can also cause hypospadias if altered [4].

The AR plays a significant role in male sexual differentiation by mediating the biological effects of gonadal androgens. The first protein coding exon in the AR gene harbours two polymorphic regions, a CAG repeat region, encoding a poly-glutamine repeat and a GGC/GGA repeat region encoding a poly-glycine repeat. Both repeats are polymorphic. Functional studies with different CAG repeat numbers have indicated an inverse relationship between the CAG repeat length and the expression level of the AR gene [9]. Expansion in the number of repeats has been associated with decreased AR transcription factor activity and as a result increased risk of impaired virility, reduced sperm production, and testicular atrophy causing infertility [10]. Significant extension of the CAG-repeat length has been reported in patients with moderate to severe under masculinisation, including micropenis with hypospadias or genital ambiguity [11]. Significant CAG-repeat extensions to ≥38 repeats is the cause of spinal-bulbar muscular atrophy. There are various AR gene mutations found in partial or complete androgen insensitivity syndrome, most of them point mutations [12].

More than 200 identified syndromes (Smith-Lemli-Opitz syndrome as the most common) and chromosomal abnormalities including gonosomal mosaicisms and autosomal deletions have been identified to be associated with the incidence of hypospadias [4].

In some boys with hypospadias several studies reported reduced response to a testosterone increase after hCG stimulation [13, 14]. Even if most boys with hypospadias show normal testosterone levels after birth, this does not necessarily imply normal androgen production in utero. Even though the use of an oral contraceptive does not lead to hypospadias, maternal exposure during early pregnancy to other oestrogenic compounds or to progestins might increase the risk of hypospadias [15, 16].

Mechanistically, genetic alterations in any of the genes involved in development of the male urogenital system could result in hypospadias. However, currently only a small percentage of hypospadias has been linked to genetic or chromosomal damage [17, 18] and several authors concluded that AR gene mutations are rarely associated with hypospadias [19, 20]. In the study of Bentvelsen et al. (1995) [17], AR levels were similar in foreskin samples of hypospadias compared with controls. While, Qiao et al. (2012) [21] noticed higher AR expression levels in boys with severe hypospadias than those with mild hypospadias and the control group. The trial by Qiao et al. seems to be one of the first showing an association between AR expression levels and the severity of hypospadias. Additionally, Kim et al. (2002) [22] confirmed in the developing human fetal penis and urethra, a greater AR expression along the ventral aspect of the glandular urethra than along the dorsal aspect of the urethral epithelium, concluding that androgens and the AR are essential for the formation of the ventral portion of the urethra and any abnormality can result in a hypospadias. For the results of the present study, further trials are needed to confirm a possible correlation between the severity of hypospadias and AR mRNA and AR protein levels.

The regulation of gene expression with gene transcription requires the attachment of hormone to the AR resulting in a conformational change with the dissociation of heat-shock proteins, transport from the cytosol into the cell nucleus, and dimerization. The AR dimer binds to a specific sequence of DNA known as a hormone-response element. The AR interacts with other proteins in the nucleus, resulting in up- or down-regulation of specific gene transcription. Up-regulation or activation of transcription results in increased synthesis of mRNA, which, in turn, is translated by ribosomes to produce specific proteins [23]. Thereby variations can appear in different areas, e.g. hormone defect, AR defect, defect of hormone-receptor binding and defect in receptor-DNA attachment. Different proteins are needed for AR function. The FK506 binding protein 4 (also known as FKBP52), for example, is a component of AR complexes, enhancing AR-mediated transactivation [24]. No differences in FK506 binding protein 4 expression and no mutations were noted between patients with hypospadias and controls [25]. However, the proven elevation of AR mRNA and AR protein in the prepuce specimens of boys with hypospadias seems to be an indirect allusion to decreased AR DNA binding capability and thus implying a signalling defect as a possible cause of hypospadias indicating further missing polypeptide encoding. To establish if differential expression of AR mRNA and AR protein can indicate the extent of the AR signalling defect and severity of hypospadias, further trials with structural analysis of the AR to corroborate this hypothesis must be carried out.

The suggestion of a possible signalling defect was provided by the study of Bell et al. (2009) [26]. In that trial nuclear translocation of the AR was intact. However, the subnuclear organisation of the AR into a hyperspeckled pattern may be affected. Thus, AR transcriptional activity may be impaired in a subgroup of patients with isolated hypospadias, and higher doses of ligand may overcome the AR signalling defect. This defect could be overcome with increasing doses of androgen in vitro. This mechanism could explain the positive effects of treatment with transdermal dihydrotestosterone gel before surgery. Kaya et al. (2008) [27] examined the efficiency of this treatment and reported that scar formations after hypospadias repair and reoperation rates were significantly lower. This benefit might be explained by the angiogenesis effects of dihydrotestosterone and by overcoming the existing signalling defects. As the secondary effects of transdermal dihydrotestosterone gel are minimal and only complaints of irritating symptoms occur, this treatment could be applied comprehensively for all grades of hypospadias. Also the results of Koff and Javanthi (1999) [28] could imply on a signalling defect. They found that treatment with hCG caused the disproportional growth of the hypospadiac phallus, turning the meatus distally, as well as decreasing the severity of chordee in all cases. Selected patients could benefit from this treatment.

Agras et al. (2006) [29] evaluated the influence of exogenous oestrogen. Their quantitative rtPCR results indicate that developing females express higher levels of AR than males throughout ontogeny. Additionally, AR mRNA expression in the genital tubercle increases in response to in utero oestrogen exposure with oestrogen-treated females having the highest AR mRNA expression, followed by oestrogen-treated males with hypospadias. These findings could be the result of relatively lower levels of androgens in female genital tubercles, indicating that the organism is trying to compensate for lower levels of androgens by increasing AR mRNA expression. The organism responded by up-regulating AR expression, so that it could respond better to any androgen that might have still been present.

In conclusion, the present data show that elevated AR mRNA and protein levels can be considered as a biochemical response to an AR signalling defect. The obvious limitation of the present study is the few children with heterogeneous characteristics (differences in age and severity of hypospadias), which subsequently restricts the interpretation of the results. Additional trials, with a larger homogeneous cohort of boys is needed to: (i) perform structural analysis of the AR, (ii) confirm a possible correlation between the severity of hypospadias/age differences and AR mRNA and AR protein levels and (iii) corroborate these preliminary findings.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of Interest
  9. References

We thank Elisabeth Richter for their support and their contribution to this work. We also want to sincerely thank each and every reviewer who dedicated his or her time and expertise to reviewing our manuscripts and everybody who supported us and collaborated with us.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of Interest
  9. References