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Downregulation of androgen, estrogen and progesterone receptor genes and protein is involved in aging-related erectile dysfunction. M Shirai1, M Yamanaka1, ...
International Journal of Impotence Research (2003) 15, 391–396 & 2003 Nature Publishing Group All rights reserved 0955-9930/03 $25.00 www.nature.com/ijir

Downregulation of androgen, estrogen and progesterone receptor genes and protein is involved in aging-related erectile dysfunction M Shirai1, M Yamanaka1, H Shiina1, M Igawa2, M Fujime3, TF Lue4 and R Dahiya1* 1

VA Medical Center and UCSF, Urology, San Francisco, California USA; 2Shimane Medical University, Izumo, Shimane, Japan; 3Juntendo University, Bunkyo-ku, Tokyo, Japan; and 4UCSF/Mt. Zion Medical Center, Department of Urology, San Francisco, California, USA

We hypothesize that downregulation of sex hormone receptors (androgen, estrogen and progesterone receptors) is involved in aging-related erectile dysfunction. To test this hypothesis, we investigated the expression of sex hormone receptors in penile crura of aging rats. A total of 40 rats were divided into four groups based on age (6, 12, 18 and 24 months), and the erectile function was analyzed by the measurement of intracavernous pressure. Gene and protein expressions of sex hormone receptors were analyzed by RT-PCR and immunostaining, respectively. The mean intracavernous pressures of 6-, 12-, 18- and 24-month-old rats were 110.1, 89.6, 73.5 and 42.7 cmH2O, respectively. Gene and protein expressions for androgen receptor, estrogen receptorbeta and progesterone receptor were present in similar levels in 6-, 12- and 18-month-old rat crura, but significantly lower or absent in 24-month-old crura. This is the first study to demonstrate that downregulation of sex hormone receptors in aging rat crura is associated with erectile dysfunction. International Journal of Impotence Research (2003) 15, 391–396. doi:10.1038/sj.ijir.3901050 Keywords: steroid hormone receptor genes; erectile dysfunction; aging

Introduction Erectile dysfunction (ED) is common in elderly men.1,2 The Massachusetts Male Aging Study reported that the combined prevalence of minimal, moderate and complete ED reached up to 52% in aging men. The prevalence of complete ED tripled from 5 to 15% in subjects aged 40–70 y old.1 Older men are more frequently affected by systemic diseases and often take a lot of medication. Although it might be possible that these factors are inter-related with the higher incidence of ED, the aging process itself undoubtedly plays a significant role in the development of ED. Circulating androgen in serum is considered to be one of the principal factors that affect male sexual function. It has been shown in rats that an adequate testosterone level is essential to maintain the availability of nitric oxide in the cavernous compartment through stimulation of expression and activity of penile endothelial and neuronal nitric oxide synthase (NOS) isoforms.3 Estrogens

and progesterone are present in the circulation in men, and it may be possible that the altered balance between sex hormones and their receptors exerts an influence on male sexual function. In the central nervous system, progesterone could mediate male sexual behavior by interacting with progesterone receptor (PR) located in the olfactory bulb.4 Although male organs produce estrogens and progesterone, their effects on penis are not clear. In a previous study, we have demonstrated that the estrogen receptor-beta (ER-b) gene expression was significantly reduced in diabetic rat penis as compared to controls.5 However, there have been few reports on the relationship of sex hormone receptors (SHR) with aging-related ED. We hypothesize that SHR (androgen receptor (AR), ER and PR) play a significant role in maintaining the male sexual function and altered levels of SHR expression are involved in the etiology of aging-related ED. To test this hypothesis, we analyzed gene and protein expressions of SHR in aging rat crura and correlated them with erectile function.

Materials and methods *Correspondence: R Dahiya, VA Medical Center and UCSF, Urology, 112F, 4150 Clement Street, San Francisco, CA 94121, USA. E-mail: [email protected] Received 26 October 2002; revised 7 March 2003; accepted 11 March 2003

Experimental animals A total of 40 male Fisher rats, divided into four groups (n ¼ 10) based on age (6, 12, 18 and 24

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months) were used in this study. These rats were purchased from the National Institute of Aging, Washington, D.C. and/or Simonson, Gilroy, California.

Electrostimulation After anesthetizing by intraperitoneal injection of pentobarbital (40 mg/kg), a lower abdominal incision was made and the cavernous nerve was exposed. Electrostimulation was performed using a delicate stainless-steel bipolar hook electrode. The diameter of each pole was 0.2 mm and the two poles were separated by 1-mm distance. Monophasic rectangular voltage pulses were generated by a computer and converted to current pulses by a computer-assisted stimulator. The stimulus parameters were 1.5 mA, 20 Hz frequency, pulse width 0.2 ms and duration 60 s. Each cavernous nerve was stimulated. For monitoring intracavernous pressure (ICP), the skin overlying the penis was incised and the penile crura were exposed. A 25-gauge needle filled with heparin solution (1000 U/ml) was inserted into the right crus and connected to a pressure monitor with polyethylene tubing.6 ICP was measured and recorded using a computer with Lab View 5.01 software (National Instruments, Austin, TX, USA). The ICP was defined as the maximum pressure obtained by the stimulation minus the basal pressure before the stimulation.

Tissue preparation Penile crura of each rat were collected immediately after completing electrostimulation. Half of the sample was fixed in 10% buffered formalin (pH 7.0) and embedded in paraffin wax, and used for immunostaining. The remaining half of each sample was immediately frozen and stored at –801C until analyzed.

Reverse transcription-polymerase chain reaction (RT-PCR) Total RNA was extracted from the samples of penile crura using Tri-Reagent (Molecular Research Center, Cincinnati, OH, USA). Complementary DNA (cDNA) was constructed by reverse transcription (Promega Corp., Madison, WI, USA) using the total RNA as a template. The samples then underwent PCR amplification, cDNA samples were diluted in 20 ml of solution containing 200 mM of dNTP, 500 nM of each primer, 0.5 U of Red Taq DNA polymerase (Sigma, St International Journal of Impotence Research

Louis, MO, USA) and PCR reaction buffer provided by the manufacturer. The primers used for rat AR, ER-a, ER-b, PR and glyceraldehyde 3-phosphate dehydrogenase (G3PDH) as a reference gene in RT-PCR were as follows: AR forward, 50 -TGTTATC TAGTCTCAACGAGC-30 ; AR reverse, 50 -CATCATTT CAGGAAAGTCC-30 ; ER-a forward, 50 -GTCCAATTC TGACAATCG-30 ; ER-a reverse, 50 -CTTCAACATTC TCCCTCC-30 ; ER-b forward, 50 -TGTACCATAGACA AGAACC-3; ER-b reverse, 50 -GGAGTATCTCTGTGT GAAG-30 ; PR forward, 50 -CTCCTGGATGAGCCTGA TG-30 ; PR reverse, 50 -CCCGAATATAGCTTGACCTC30 , G3PDH forward, 50 -TCCCATCACCATCTTCCA-30 and G3PDH reverse, 50 -CATCACGCCACAGTTTCC30 . PCR reactions were performed in a PTC-200 thermal cycler (MJ Research, Watertown, MA, USA) at 941C for 3 min; 32 cycles at 941C for 1 min, 521C for 1 min, 721C for 1 min; followed by final extension at 721C for 5 min. For semiquantitative analysis, three other dilution sets of each sample (original, 1/ 2, 1/4 and 1/8 dilution) underwent PCR. Annealing temperature was 521C for all RT-PCR reactions. The PCR products were electrophoresed on 2.0% agarose gel and the expression level of these genes was evaluated by an ImageJ software (http://rsb.info. nih.gov/ij), and the areas under the curves were calculated and analyzed. PCR cycles were adjusted until these four preparations in each sample were within linear range. Between 28 and 32 cycles all SHR PCR reactions were within linear range. The expression of each gene was quantified relative to G3PDH expression and expressed as arbitrary units (AU). The expected sizes of PCR products were 587 base pair (bp), 346, 633, 293 and 380 bp in AR, ER-a, ER-b, PR and G3PDH, respectively.

Immunohistochemistry Tissue sections were cut (4 mm) and mounted on a slide. Tissue block sections were dewaxed, rehydrated, incubated with 0.3%(v/v) hydrogen peroxide for 20 min, and autoclaved at 1211C for 15 min in 10 mM citrate buffer (pH 6.0). The samples were incubated with serum block (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) for 20 min at room temperature followed by overnight at 41C with the following antibodies: 1:150 dilution of a monoclonal antibody against mouse ER-a (Ab-10) (Lab Vision Corp., Fremont, CA, USA), 1:400 dilution of a polyclonal antibody against rabbit AR (C-19), 1:400 dilution of a polyclonal antibody against rabbit ER-b (H-150) and 1:100 dilution of a polyclonal antibody against rabbit PR (H-190) (Santa Cruz Biotechnology, Inc.). After the slides were washed, they were incubated with a biotinylated goat anti-mouse antibody and streptavidin-peroxidase (Lab Vision Corp.) for ER-a; or biotinylated goat anti-rabbit antibody and HRP-streptavidin (Santa Cruz Biotechnology,

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Inc.) for AR, ER-b and PR at room temperature for 30 min. Following incubation with the avidin–biotin complex, the antigen–antibody complexes were visualized with diaminobenzidine (Lab Vision Corp.). The sections were counterstained using hematoxylin. As a negative control, normal goat IgG was substituted for the specific antibody at the same dilution. Paraffin sections of a normal rat uterus and prostate were used as a positive control. A Leitz LaboLux S-microscope and a Nikon digital camera were used to determine each SHR immunoreactivity within the cavernous tissue. The immunostaining pictures were analyzed on a PC with Image Pro software. The nucleus in the cavernous tissue was identified at  400. The pixels in the color range that corresponded to the stain of each SHR and negative nucleus were counted and expressed as the percentage of the total number of pixels in the field. The number of pixels that corresponded to all nucleus (positive and negative nuclei) was counted, and defined it as 100% in each area. Three different sections in each animal were used for each SHR immunostaining, and 10 different fields were analyzed in each specimen.

Gene expression of AR, ER-a, ER-b and PR in aging rat crura (Figures 1 and 2).

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Typical results and summarized data of each RTPCR are shown in Figures 1 and 2, respectively. There was no significant difference in AR mRNA expression between 6-, 12- and 18-month-old rats. In 24-month-old rats, expression of AR mRNA transcript was significantly reduced (Po0.01, respectively). For ER-a mRNA, there were two splicing isoforms within the primer spanning region, and a strong band corresponded to 346 bp (Figure 1). There was no significant difference in the expression of ER-a mRNA among rats of different ages. As shown in Figure 1, expression level of the splicing variant of ER-a mRNA was decreased with advancing age.

Statistical analysis Parametric data were expressed as mean7s.e. Data were analyzed by one-way analysis of variance (ANOVA) with Fisher’s protected least-significance t post hoc test using StatView 5.01 software (SAS Institute Inc., Cary, NC, USA). The P-value of less than 0.05 was considered to be statistically significant.

Results Functional study of aging rat penis The ICP of rats after electrostimulation was reduced with advancing age. In 6-month-old rat, the pattern of ICP curve was sharp and the peak pressure was the highest. On the other hand, in 24-month-old rat, the peak pressure was the lowest. The mean ICP was 110.178.3, 89.6712.1, 73.5713.9 and 42.77 13.3 cm H2O in 6-, 12-, 18- and 24-month-old rats, respectively, indicating that ICP was decreased with advancing age. Significant differences in ICP were observed between 6- and 12-month-old rats, or between 18- and 24-month-old rats (Po0.05, respectively). The difference in ICP between 12- and 18month-old rats was close to statistical significance (P ¼ 0.05).

Figure 1 Typical RT-PCR results of SHR in aging rats are shown. Note that in 24-month-old rat, expression level of AR and ER-b messenger RNA transcript is weak but preserved, while that of PR is lost (* ¼ 100 bp DNA ladder). Relative mRNA value is shown on each band.

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Figure 3 Typical immunostainings of AR (a and c), ER-a (b and d). Note that protein expression of AR was decreased in 24-monthold rat, whereas that ER-a was not significantly different: (a and b) 6-month (  400); (c and d): 24-month (  400).

Figure 2 Alterations of SHR mRNA expression with aging are shown. Expression of AR, ER-b and PR mRNA was decreased along with aging, whereas that of ER-a was not significantly different among all aging groups. Values are expressed as means7s.e. (AU ¼ arbitrary units).

ER-b mRNA expression among 6-, 12- and 18month-old rats did not differ, but was significantly reduced in 24-month-old rats (Po0.01, respectively). The expression of PR mRNA transcript was the highest in 6-month and was lost in 24-month-old rats. The difference in PR mRNA expression between 6- and 24-month-old rats reached statistical significance (Po0.01), but no significant difference was observed between 12- and 18-month-old rats.

Protein expression of AR, ER-a, ER-b and PR in aging rat crura (Figures 3–5). Typical result of each immunostaining is shown in Figures 3 and 4, and summarized data are shown in Figure 5. AR protein expression was the highest in 6month-old rat. The immunoreactivity was observed in the nuclei of smooth muscle cells of small artery as well as smooth muscle fibers within the crura (Figure 3a). In 24-month-old rat crura, the number of AR-positive cells was the lowest as compared to 6month-old rats (Figure 3c). A significant difference was observed between 12- and 18-month-old rats or 18- and 24-month-old rats (Po0.05); however, the difference between 6- and 12-month-old rats did not reach statistical significance. International Journal of Impotence Research

Figure 4 Typical immunostainings of ER-b (a and c) and PR (b and d). Note that protein expression of ER-b and PR was decreased and lost in 24-month-old rat, respectively: (a and b) 6-month (  400); (c and d) 24-month (  400).

ER-a-positive cells were distributed in the nuclei of smooth muscle cells in 6-month-old rat crura (Figure 3b). The number of the positive cells was similar among 6- and 24-month-old groups (Figures 3b and d) and no significant difference in ER-a immunoreactivity was observed among rats of different ages. ER-b immunoreactivity was reduced with advancing age of rats (Figure 4a and c). The difference in the immunoreactivity reached statistical significance between 6- and 12-month as well as between 18- and 24-month-old rats (both Po0.05). PR immunoreactivity was significantly reduced with advancing age (Figure 4b and d). Interestingly in 24-month-old rat crura, the PR immunoreactivity was completely lost (Figure 4d).

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Figure 5 Alterations of SHR immunoreactivity with aging. Reduction of immunoreactivity of AR, ER-b and PR was associated with aging, while ER-a immunoreactivity was not significantly changed among all age groups. In 24-month-old rats, AR and ER-b protein expressions were significantly lower than 6-, 12- and 18-month-old rats (Po0.05). PR protein expression was lost in 24-month-old rats. Values are expressed as means7s.e.

Discussion Previous studies in our laboratory and in others have shown that aging is associated with ED.7,8 The molecular mechanisms of aging-related ED are not fully understood. We hypothesized that alterations in SHR are involved in the pathogenesis of ED in aging rats. To test this hypothesis, we analyzed gene and protein expression of AR, ER and PR in aging rat crura. First, we measured ICP to verify the presence of ED in the aging rat. ICP was lowered with advanced age, which is consistent with the finding observed in aging ED.9 Several lines of studies have demonstrated that androgen deprivation is related to apoptosis of smooth muscle cells in addition to an increase in connective tissue contents.10–12 Penile eNOS and nNOS are two major contributors to maintain erectile function, of which activity is restored after androgen stimulation in castrated rats. In the present study, both levels of protein and mRNA transcript of AR were significantly decreased with advancing age in rat crura. In addition, this agingrelated alteration of AR expression was well correlated with reduction of ICP. Circulating level of serum testosterone has been reported to be reduced with advancing age in rat.13 Even if sufficient amount of androgen is present in the older rats, significant reduction of AR expression itself might

cause relative androgen deprivation status in aging rat crura. These alterations are probably associated with the enhancement of degenerative processes of smooth muscle cells and/or reduction in eNOS and nNOS.3,14 Estrogen functions as regulator of growth and differentiation in various target tissues. Its receptors, ER-a and ER-b mediate the functional role of estrogen in target organs, but the distribution of ERs is quite different among rat tissues. The pituitary, kidney, epididymis and adrenal are predominant for ER-a, whereas prostate, ovary, lung, bladder, brain and testis are higher in ER-b.15 In rat crura, gene and protein expression of ER-b was reduced with advancing age and markedly decreased in 24-month-old rats, whereas ER-a gene and protein expressions were not significantly changed among 6-, 12-, 18- and 24-month-old rats. In addition, the age-related ICP alteration correlated with the change of ER-b expression, but not with that of ER-a expression. These findings indicate that the functionally predominant form of ER in rat crura is ER-b and age-related alteration of ER-b is probably related to the pathogenesis of ED in older rats. The mechanism of ER-b involved in aging-related ED still remains to be elucidated. Estrogen exerts direct vascular protection effect on endothelial and/or smooth muscle cells, and this effect is exclusively mediated by ER-b, but not by ER-a.16,17 Estrogen also exerts an inhibitory effect on apoptosis in human endothelial cells with upregulation of antiapoptotic Bcl-2.18,19 Loss of cytoprotective role of ER-b with aging might be one of the mechanisms of development and/or progression of aging-related ED. Alternatively, potential antiapoptotic effect by estrogen might be impaired by reduced expression of ER-b with advancing age. In fact, a recent publication clearly demonstrated that loss of antiapoptotic genes in rat crura is associated with the pathognesis of aging-related ED.20 There are no reports of the relationship between development and/or progression of ED and PR expression in the aging rat crura. Progesterone has been shown to be involved in the modulation of NOS activity21 and may be due to PR. The most interesting finding was that PR gene and protein expression were almost lost in 24-month-old rat crura. In addition, the mean ICP drop between 18and 24-month-old rats (about 31 cm H2O) is greater than those observed between 6- and 12-month (about 20 cm H2O) or between 12- and 18-month (about 16 cm H2O). Taken together, it might be plausible that the impaired functional role of PR as modulator of NOS activity reflects the lowest level of ICP in 24-month-old rats. In summary, ICP alteration in relation to aging crura is well correlated with AR, ER-b and PR expression. Even between 18- and 24-month of age, the abrupt reduction in AR, ER-b and PR still displayed significant correlation with ICP

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reduction. These findings strongly suggest that AR, ER-b and PR are involved in the pathogenesis of ED with advancing age.

Conclusions To our knowledge, this is the first study demonstrating that downregulation of gene and protein expression of ER-b and PR is associated with ED of aging rats. These results may be important in understanding the pathogenesis of aging-related ED and also in providing better strategies for the management of aging-related ED.

Acknowledgements This research was supported by the National Institutes of Health Grants RO1DK055040 and RO1AG016870.

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