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Expression of Estrogen Receptor Alpha and Beta Is Decreased in Hypospadias Liang Qiao,* Esequiel Rodriguez, Jr.,* Dana A

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Expression of Estrogen Receptor Alpha and Beta Is Decreased in Hypospadias Liang Qiao,* Esequiel Rodriguez, Jr.,* Dana A Expression of Estrogen Receptor Alpha and Beta is Decreased in Hypospadias Liang Qiao,* Esequiel Rodriguez, Jr.,* Dana A. Weiss, Max Ferretti, Gail Risbridger, Gerald R. Cunha and Laurence S. Baskin† From the Division of Pediatric Urology, University of California, San Francisco Children’s Hospital, San Francisco, California, and Department of Anatomy and Developmental Biology, Monash University, Clayton, Victoria, Australia (GR) ␣ ␤ Purpose: Estrogenic endocrine disruptors acting via estrogen receptors and Abbreviations have been implicated in the etiology of hypospadias. However, the expression and and Acronyms ␣ ␤ distribution of estrogen receptors and in normal and hypospadiac human ER ϭ estrogen receptor foreskins is unknown. We characterized the location and expression of estrogen ESR1 ϭ estrogen receptor alpha receptors ␣ and ␤ in normal and hypospadiac foreskins. ϭ Materials and Methods: We prospectively collected excess foreskin from 35 ESR2 estrogen receptor beta patients undergoing hypospadias repair and 15 patients undergoing elective GADPH ϭ glyceraldehyde-3- circumcision. Hypospadias was classified as severe in 18 patients and mild in 17 phosphate dehydrogenase based on the ectopic position of the meatus. mRNA expression levels in estrogen IHC ϭ immunohistochemical receptors ␣ and ␤ were quantified using reverse transcriptase polymerase chain PCR ϭ polymerase chain reaction reaction. Receptor location was characterized by immunohistochemical analysis. RT ϭ reverse transcriptase Additionally immunohistochemical analysis was performed in 4 archived human fetal penises. Ϯ Ϯ Submitted for publication August 14, 2011. Results: Mean SD ages were similar for the circumcision (9.5 3 months) and Study was approved by University of Califor- hypospadias repair groups (9 Ϯ 3 months, p ϭ 0.75). mRNA expression levels in nia, San Francisco human research committee. estrogen receptors ␣ and ␤ were significantly decreased in hypospadiac foreskin Study was supported by NIH Grant RO1 Ͻ DK0581050. cases compared to controls (p 0.001), while no statistically significant differ- * Each author contributed equally to the study. ences were seen between foreskins with severe and mild hypospadias. Estrogen † Correspondence: Division of Pediatric Urol- receptor ␤ immunostaining was strong in normal foreskin but weak in hypospa- ogy, University of California, San Francisco Chil- diac foreskin. Estrogen receptor ␤ immunoreactivity was most intense in the dren’s Hospital, San Francisco, California 94143 (e-mail: [email protected]). stratum basale and stratum spinosum. Estrogen receptor ␣ immunostaining was weak in normal and mild hypospadias foreskin, and undetectable in severe hypospadias. Fetal penises expressed strong estrogen receptor ␤ immunopositiv- ity in the urethral plate epithelium, corpus spongiosum, corpora cavernosa and penile skin, while estrogen receptor ␣ immunostaining was not detected. Conclusions: These data demonstrate a difference in estrogen receptor ␣ and ␤ expression and location in the foreskin of patients with hypospadias compared to controls. These findings are consistent with aberrant estrogenic effects having a role in the etiology of hypospadias. Key Words: foreskin; hypospadias; penile diseases; penis; receptors, estrogen HYPOSPADIAS is a common congenital dias is associated with penile curva- abnormality characterized by an ecto- ture and incomplete development of pic urethral meatus along the ventral the ventral foreskin.1,2 Hypospadias surface of the penis proximal to the affects approximately 1 in 200 to 300 normal glanular location. Hypospa- live male births,1 although recent ep- 0022-5347/12/1874-1427/0 Vol. 187, 1427-1433, April 2012 ® THE JOURNAL OF UROLOGY Printed in U.S.A. www.jurology.com 1427 © 2012 by AMERICAN UROLOGICAL ASSOCIATION EDUCATION AND RESEARCH,INC. DOI:10.1016/j.juro.2011.12.008 1428 ESTROGEN RECEPTORS ALPHA AND BETA IN PATIENTS WITH HYPOSPADIAS idemiological data suggest an increasing inci- eliminate DNA contamination by using proteinase K (1.3 dence.3–5 Although the etiology remains elusive, a mg, Roche Diagnostics GmbH, Mannheim, Germany) and possible explanation for the increasing incidence of RNase-free DNase I (20 ␮l, Qiagen, Inc., Valencia, Cali- hypospadias in industrialized countries is fetal ex- fornia). A NanoDrop® spectrophotometer was used to posure to estrogenic compounds or other environ- measure the quantity and purity of RNA. mental endocrine disruptors.4,6 Supporting this the- RT-PCR was performed according to standard protocol. Briefly 2.5 ␮g RNA were reverse transcribed in a reaction ory is the observation that male offspring of women volume of 20 ␮l and diluted with Tris-ethylenediaminetet- exposed to diethylstilbestrol during pregnancy have 7 raacetic acid buffer (10 mM Tris HCl, pH 8, 1 mM ethyl- a higher incidence of hypospadias, consumption of enediaminetetraacetic acid). PCR primers were designed phytoestrogens (such as genistein) during gestation according to the specific target gene sequence published in is associated with a higher incidence of hypospadias PubMed and were synthesized by IDT®. The RT-PCR 8 in boys, and in laboratory models neonatal expo- primers are outlined in the Appendix. SYBR® Green real- sure to estrogens and genistein causes hypospa- time quantitative PCR was performed using a 7300 fast dias.9–11 Consistent with this theory is the discovery sequence detection system (Applied Biosystems™) accord- of estrogen receptors ␣ and ␤, crucial for signal ing to the manufacture instructions. Primer titration and transduction of estrogenic substances, in the penile dissociation experiments were performed so that no tissue of rat,12 mouse13 and humans.14,15 primer dimers or false amplifications would interfere with Human skin contains ESR1 and ESR2, and is an the result. Cycle threshold number was extracted for the important estrogen responsive organ.16 Although reference (GADPH) and target genes with auto baseline foreskin may have similar characteristics, to date and manual thresholds. PCR was repeated 3 times for each sample. Expression levels of ESR1 and ESR2 are the exact location and expression of estrogen recep- reported relative to GADPH using a 2 delta-delta cycle tors have not been defined in normal or hypospadiac threshold.18 There was no difference in the amplification human foreskin. Characterizing the cellular location kinetics of GADPH and ESR1/ESR2. and expression patterns of estrogen receptors in nor- mal and hypospadiac human foreskin may expand Immunohistochemical Staining our understanding of the causes of hypospadias be- Foreskin samples were fixed in formalin, paraffin embed- ␮ yond the murine model. In this study we used real- ded and sectioned at 5 m. Antigens were retrieved with time polymerase chain reaction to qualify the mRNA antigen unmasking solution (Vector Laboratories, Inc., Burlingame, California). After blocking, slides were incu- expression of ESR1 and ESR2, and immunohisto- bated overnight at 4C with rabbit anti-ER␤ antibody (1:100 chemical staining to determine receptor location. We dilution, Upstate Biotechnology, Inc., Lake Placid, New hypothesize that the location and expression of York), and mouse anti-ER␣ antibody (1:30 dilution, cloneID5, ESR1 and ESR2 differ between normal and hypos- Dakocytomation, Carpinteria, California). Staining of the padiac foreskin. tissue was performed with the Elite ABC kit (Vector Labo- ratories, Inc.), followed by hematoxylin counterstaining. Adult mouse uterus and prostate served as positive con- MATERIALS AND METHODS trols for ER␣ and ER␤. Sections exposed to all steps except This study was approved by the University of California, application of the primary antibodies were used as nega- San Francisco human research committee. Any subject tive controls. Four human fetal penises from our tissue younger than 14 months undergoing initial hypospadias bank were also processed in a similar protocol. To confirm ␣ repair or elective circumcision at the University of Cali- our results concerning the lack of detectable ER in the fornia, San Francisco between 2005 and 2010 was eligible human fetal tissue, we repeated the analysis using a dif- ␣ for the study. All subjects were prospectively enrolled, and ferent mouse monoclonal anti-ER antibody (1:100 dilu- informed consent was obtained preoperatively from par- tion, ab2746, abcam®). Stained serial sections were exam- ents/legal guardians. Excess preputial tissue was obtained ined using a DM4000B brightfield microscope (Leica from 35 male subjects with hypospadias undergoing sur- Microsystems, Inc., Buffalo Grove, Illinois). Digital im- gical repair and 15 patients undergoing elective circumci- ages were acquired with a DFC500 camera (Leica Micro- sion. The degree of hypospadias was determined by the systems, Inc.). Once images were taken, the density of position of the ectopic urethral meatus and was classified nuclear staining was graded on a subjective scale as none as mild (at or distal to mid shaft of penis) in 17 patients (0), weak (ϩ), intermediate (ϩϩ) or strong (ϩϩϩ). Each and severe (proximal to mid shaft of penis) in 18. Four determination was based on the agreement of 2 observers human fetal penises were obtained from our archived who were blinded to the treatment group when inspecting tissue bank. Estimated gestational ages based on last the images. menstrual period, head circumference and femur length Statistical Analysis were approximately 8 to 10, 14, 20 and 24 weeks. The difference in mRNA expression levels of ESR1 and
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