Expression of Estrogen Receptors in Human Corpus Cavernosum and Male Urethra
Division of Gynecologic Endocrinology and Reproductive Medicine (WD,JCH), University of Vienna Medical School, Vienna, Austria; Department of Clinical Pathology (AH), University of Vienna Medical School, Vienna, Austria; and Department of Urology (WJR), Korneuburg Hospital, Korneuburg, Austria
Correspondence to: Wolf Dietrich, MD, Div. of Gynecologic Endocrinology and Reproductive Medicine, Univ. of Vienna Medical School, Waehringer Guertel 1820, 1090 Vienna, Austria. E-mail: wolf.dietrich{at}akh-wien.ac.at
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Summary |
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Key Words: estrogen receptor- estrogen receptor-ß androgen receptor corpus cavernosum endothelium urethra
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Introduction |
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Estrogens are known to stimulate growth, blood flow, and water retention in sexual organs (Jenner et al. 1972; Batra et al. 1985
). There, and in the lower urinary tract, ER
and ERß have been described in testis, prostate, and efferent ducts (Bodker et al. 1999
; Makela et al. 2000
; Salmi et al. 2001
). Most of these studies have been performed in rodents, but there is only one piece of evidence of ERs in the penis (rat; Jesmin et al. 2002
). The goal of our in vitro study was to prove the evidence of the two ER isoforms, as well as of the androgen receptor (AR) and the progesterone receptor (PGR), in human corpus cavernosum and male urethral tissue, exploring the protein expression of these receptors by immunohistochemistry (IHC). The results represent the first report that ER
and particularly ERß are regularly expressed in human penile tissue.
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Materials and Methods |
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Immunohistochemistry
Four-µm-thick sections of the tumor-free resection edge were dewaxed and rehydrated. Deparaffinization was carried out in xylene and graded ethanols. Endogenous peroxidase was blocked by incubation in 5% H2O2 in methanol for 15 min. Heat-mediated antigen retrieval was performed by microwaving three times for 5 min at 500 W in Citra-Plus solution (BioGenex; San Ramon, CA) for ERß staining or citrate buffer (pH 6.0) for all others. Unspecific bindings were blocked using the biotin blocking system (DAKO; Carpinteria, CA). Incubation was carried out with the following antibodies in a humidified chamber:
Antibodies against AR, ER, and PGR have been well characterized by Western blotting on human recombinant receptors (detailed documentation at the manufacturer, DAKO) and are widely used in clinical routine diagnostics. The antibody raised against ERß has been characterized by absorption studies (Skliris et al. 2002
). These authors found it most suitable for IHC on paraffin-embedded tissue compared to six other ERß antibodies. To further test the specificity of 1D5 and 14C8, antibodies were diluted to working concentration and preabsorbed with a 100-fold excess of the respective antigens, i.e., recombinant human ER
-protein (Affinity BioReagents; Golden, CO) at 23 pmol/100 µl and recombinant human ERß-protein (Alexis Biochemicals, San Diego, CA) at 44 pmol/100 µl for 1 hr at RT. Solutions were centrifuged at 13,000 rpm for 10 min and the supernatant was used for incubation (see Figure 2
).
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IHC was performed semiquantitatively on three sections per sample and antibody.
The intensity of nuclear staining and the number of stained cells were examined independently by two investigators (WD and AH). No significant staining was scored as 0, scattered or weak immunostaining as +, and intense immunostaining in approximately 50% or more of respective cells as ++ (Fan et al. 2002).
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Results |
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Urethra
Urethral epithelium showed strong nuclear expression of AR, predominantly in the basal cell layer. Immunostaining decreased in the luminal direction to negative in the superficial cells. ER showed strongest positivity in the intermediate cells of all urethras explored, but staining was weaker than that of AR. Further, ERß was highly expressed in almost all nuclei, and, much more weakly, in the cytoplasm of epithelial cells of every urethra explored.
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Discussion |
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The relevance of androgens and other sex hormones, particularly estrogen, in the modulation of the structure and function of the urothelium and the distal urethra is not yet understood. Although two reports also described strong reactivity of AR in developing human and adult rat urethra by IHC (Salmi et al. 2001; Kim et al. 2002
), there is no evidence in adult human male urethra. In our study, the basal cells markedly expressed AR, indicating that androgen is involved in the growth of human male urothelium. Whereas in the male rat urethra, Makela et al. (2000)
could not detect ER
by in situ hybridization and IHC, a more recent study found weak ER
protein expression in the proximal and distal female urethra (Blakeman et al. 2000
). However, the importance of moderate expression of ER
in the suprabasal layer of male urothelium remains unclear, particularly because ERß, which is known to exert inverse transcriptional effects, is highly expressed in almost all epithelial cells. Here, our results showing strong expression in nuclei of the epithelial cells and, additionally, moderate cytoplasmic expression are in agreement with all previous studies in male rat urethras.
In view of the observation that ERß is expressed in human penile vasculature and urothelium in excess to the other sex steroid receptors, we propose that estrogen could play a significant role in penile blood vessel regulation and epithelial function, operating preferably on the ERß-isoform. It can be suggested that estrogen's action supports resistance to increased mechanical and hemodynamic stress, according to Lindner et al. (1998), and maintains vascular tone and perhaps erectile function. Further studies will be needed to discover estrogen-regulated genes, especially those being controlled by ERß, in the male reproductive system, and to clarify the possible involvement of aromatization of androgens to estrogen in penile tissue.
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Footnotes |
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Literature Cited |
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