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From the Department of Reproduction and Genetics, Nanjing University School of Clinical Medicine, Jinling Hospital, Nanjing, Jiangsu, China.
| Correspondence to: Dr Yufeng Huang, 305 East Zhongshan Road, Nanjing 210002, PR China (e-mail: pljandrol{at}163.com). |
| Received for publication May 22, 2007; accepted for publication July 30, 2007. |
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Abstract |
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and β) in the
corpora cavernosa were also detected immunohistochemically. When the rats were
examined at adulthood, we observed that those animals treated with a medium
(20 mg/kg) or high (100 mg/kg) dose of daidzein, but not with a low dose (2
mg/kg), showed lower plasma testosterone levels and attenuated erectile
parameters, including apomorphine-induced erections and intracavernous
pressure concomitant with markedly decreased expression of estrogen receptor
β in the corpora cavernosa. However, the penis still grew to its normal
size, as in controls. Thus, these results suggested that exposure of juvenile
rats to daidzein in a relatively large amount could adversely affect penile
erection in adulthood.
Key words: Isoflavones, estrogens, apomorphine-induced erections, penis, animal model
Phytoestrogens can bind to the estrogen receptor (ER), particularly ERβ, and induce estrogen-like effects in animals, humans, and cells in culture (Collins et al, 1997; Usui, 2006). Although most of these compounds are rather weak estrogens in comparison with estradiol or potent synthetic estrogens, phytoestrogen exposure, at sufficiently high dietary levels, may result in biologic responses in humans and animals, with favorable as well as unfavorable consequences (Jefferson and Newbold, 2000). Recently ER was detected in the penis (Mowa et al, 2006), and it is believed that ER may be involved in erectile dysfunction (Srilatha and Adaikan, 2004). On the other hand, it has been reported that estradiol might play a role in causation and/or persistence of erectile dysfunction in patients with low testosterone levels (Srilatha et al, 2007). Furthermore, epidemiologic surveys demonstrated that erectile dysfunction incidence in men aged more than 20 years was nearly 10% higher in Chinese than in Americans (28.3% vs 18.4%) (Bai et al, 2004; Selvin et al, 2007). In consideration of the information above, there might be an association between impotence and dietary intake of phytoestrogens in Asians who typically consume much more soybeans and related products than Americans. However, there are few reports in the literature regarding the effects of phytoestrogens on penile erection (Srilatha and Adaikan, 2004). In our previous research, we observed that exposure of adult rats to daidzein could attenuate apomorphine-induced erections (unpublished data). The aim of the present study was to investigate the impact of daidzein on erectile function when rats are exposed in early life. To the best of our knowledge, this is the first study to evaluate the effect of daidzein exposure in early life on erectile capacity.
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Methods |
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98% as determined by high-performance liquid
chromatography) was obtained from Nanjing Rally Biochemical Inc (Nanjing,
China). Apomorphine and diethylstilbestrol (DES) were obtained from
Sigma-Aldrich (St Louis, Mo). Radioimmunoassay (RIA) kits for the detection of
testosterone were obtained from Beijing Furui Bio-engineering Co (Beijing,
China). Enzyme linked immunosorbent assay (ELISA) kits for the detection of
luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were obtained
from Adlitteram Diagnostic Laboratories, Inc (San Diego, Calif). Mouse
monoclonal anti-ER antibody and rabbit polyclonal anti-ERβ
antibody were obtained from Abcam (Cambridge, United Kingdom).
Peroxidase-labeled goat anti-mouse IgG and goat anti-rabbit IgG antibodies
were obtained from Kirkegaard & Perry Laboratories, Inc (Gaithersburg,
Md).
Animals
Prior to study initiation, the experimental protocol was reviewed and
approved by the Jinling Hospital Ethical Committee to Animal Care and Use.
Forty male juvenile Sprague-Dawley rats of 28 to 32 days weighing 140 to 170 g
were obtained from the Laboratory Animal Center of Jinling Hospital. They were
randomly assigned based on weight into 5 groups of 8 animals each. Room
temperature was maintained at 20°C ± 1°C under a 12-hour
light/dark cycle with lights off at 1800 hours. The animals had free access to
tap water and pelleted commercial rodent chow without soybean or alfalfa
(Jiangsu Xietong Organism Engineering Co Ltd, Nanjing, China). Body weights
were measured each week during the experiment.
Treatments
Each group was gavaged daily for 90 days with 1 mL of distilled water
containing daidzein at a dose of 2 mg/kg (low-dose group), 20 mg/kg
(medium-dose group), 100 mg/kg (high-dose group), 1 mL of distilled water
containing DES at a dose of 0.1 mg/kg (positive-control group; this dosage of
DES is known to cause erectile dysfunction in rats
[Adaikan and Srilatha, 2003]),
or distilled water only (control group). Dosages were periodically adjusted
based upon the weights of the animals.
In Vivo Penile Erection
At the end of the study when the animals reached adulthood, the
apomorphine-induced penile erection test established by Heaton et al
(1991) was performed. Animals
were allowed to adapt to the dark, soundproof testing room for 1 hour before
the start of the experiment. Apomorphine was freshly prepared with 0.1%
ascorbic acid and injected subcutaneously into the napes of the necks of rats
at a dose of 80 µg/kg. After the drug injection, each rat was placed
immediately into a transparent Plexiglas cage (30 x 25 x 25 cm).
Thirty-minute observations started after the rats were placed in cages. Penile
erections were considered to occur when all of the following behaviors
presented: the animal stood upright, bent its head down to reach the genital
area, held its engorged penis by its forepaws, and licked its glans penis. We
observed and counted penile erection episodes during the 30-minute observation
period. We measured the latency of the first erection and the number of
erections per animal. The latency was recorded as 30 minutes if no erection
occurred during the observation period.
Electrostimulation for Penile Erection
The penile erection was also assessed by measuring the intracavernous
pressure following electrostimulation of cavernous nerves. Considering that
estrogen and daidzein have apparent impacts on blood pressure in rats
(Peng et al, 2003;
Cao et al, 2006), we did not
monitor mean arterial pressure to calculate the intracavernous pressure:mean
arterial pressure ratio. Under general anesthesia with sodium pentobarbital
(50 mg/kg) intraperitoneally, each animal was placed in a supine position. A
lower abdominal incision was made, the skin overlying the penis was incised,
and the penile crura exposed. The testes were retracted, and the scrotum was
divided and packed into the upper abdomen along loops of the bowel. The
cavernous nerve, which was seen on the postlateral surface of the prostate
arising from the major pelvic ganglion, was exposed. For monitoring the
intracavernous pressure, a 27-gauge needle was inserted into the right crura
and connected to a pressure monitor. Electrostimulation was performed using a
delicate stainless steel bipolar hook electrode. The magnitude of electrical
voltage was 5.0 V with a 20-Hz frequency, pulse width of 2 milliseconds, and
duration of 60 seconds. Intracavernous pressure was measured and recorded with
LabView 5.01 software (NI Corp, Austin, Tex). The intracavernous pressure was
defined as the maximal pressure obtained by stimulation minus the basal
pressure before stimulation.
Blood Sampling
After intracavernous pressure monitoring, 3.0 mL of blood was collected via
heart from each animal. The plasma was prepared from the blood and stored at
-20°C until assayed.
Determination of Sex Hormone Concentrations
Plasma concentrations of testosterone were determined in duplicate using a
double-antibody RIA. The average intra-assay and interassay coefficients of
variation (CV) were less than 9.5% and 10.3%, respectively, with an average
sensitivity of 0.1 ng/mL. LH and FSH concentrations in plasma were determined
in duplicate using a competitive ELISA
(Pappa et al, 1999). The
average intra-assay and interassay CV were 4.7% and 6.8%, respectively, with
an average sensitivity of 0.1 ng/mL for LH. For FSH, the average intra-assay
and interassay CV were less than 5.5% and less than 6.4%, respectively, with
an average sensitivity of 0.1 ng/mL.
Examination of the Penis and Tissue Process
Animals were sacrificed, and each penis was grossly examined for its
length, diameter, and weight. The stretched length was measured from the tip
of the glans penis to the midpoint of the ischial arch (the point of origin of
the root of the penis) and the diameter from the middle of the penile shaft
with a caliper (calibrations up to 0.1 mm). After the free, loose connective
tissue was removed, the entire penis was weighed. For each subject, the penile
index was calculated as penile weight (mg) divided by body weight (g). The
middle of the penile body was cut and then immediately fixed in 4%
paraformaldehyde in 0.1 mol/L phosphate-buffered saline (PBS; pH 7.4). One
sample was obtained from all tissues and blocked with paraffin for routine
follow-up procedures. Consecutive sections with thicknesses of 4 µm were
prepared. Anti-ER and anti-ERβ antibodies were used
immunohistochemically to visualize ER and ERβ.
Immunohistochemistry Staining Procedure and Analysis
After deparaffinization and rehydration, the sections were washed in PBS
for 5 minutes. All sections were treated with citrate buffer in a microwave
(700 W) for 5 minutes for antigen retrieval. Quenching of endogenous
peroxidase activity was achieved by incubating the sections for 20 minutes in
3% hydrogen peroxide. After washing in PBS, the sections were incubated with
blocking serum for 20 minutes. Thereafter, the sections were incubated with
the primary anti-ER (1:100) or anti-ERβ (1:800) antibody for 2
hours at 37°C and then rinsed in PBS and incubated with the secondary
antibody, peroxidase-labeled goat anti-mouse antibody (1:100) for
anti-ER or goat anti-rabbit antibody (1:500) for anti-ERβ for 30
minutes. After washing in PBS, they were exposed for 10 minutes to 0.1%
diaminobenzidine (Boster, Wuhan, China) and 0.2% hydrogen peroxide in 50
mmol/L Tris buffer (pH 8). Mayer hematoxylin stain was used as the background.
Sections were covered with neutral gum. Negative control sections had PBS
added instead of primary antibody, with all other steps the same.
The immunolabeling stains were evaluated under 400x magnification. The intensity of the staining in corpora cavernosa was examined independently by 2 observers who were blind to the animal group. Staining intensities were scored semiquantitatively: 0 = no staining; 1 = weak or scattered immunostaining; 2 = moderate staining (approximately 50% of cells stained); 3 = strong staining (>50% of cells stained). For each animal, 3 sections were evaluated and the mean score was calculated.
Statistical Analysis
Results were expressed as means ± SE. The data were analyzed using
1-way analysis of variance followed by the least significant difference test
for post hoc comparison. All calculations and analyses were done using SPSS
version 13.0 software (SPSS Inc, Chicago, Ill). P < .05 was set as
significant.
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Results |
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Apomorphine-Induced Penile Erection
Daidzein-treated rats exhibited dose-related decreases in responses to
apomorphine. As illustrated in Figure 1A
and B, the mean number of penile erections decreased, whereas
erectile latencies increased in the low-dose daidzein group; however, the
differences were not statistically significant compared with controls (both
P > .05). The mean number of erections significantly decreased by
61% in the medium-dose daidzein group and by 57% in the high-dose daidzein
group; meanwhile, mean erectile latencies increased significantly. In the DES
group, the mean number of erections was reduced to 0.5 ± 0.2, which was
approximately 50% of the medium- or high-dose daidzein group value and almost
20% of the control value.
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Gross Observations of the Penis
Daidzein had no apparent effect on penile growth in exposed rats. No
significant differences between any of the daidzein groups and the controls
were seen in penile weight, length, diameter, and index. However, all but one
animal (7/8) treated with DES displayed microphallus
(Table 1).
Concentrations of Sex Hormones
As shown in Table 2, there
were no statistically significant differences in plasma testosterone, LH, and
FSH levels between the low-dose daidzein group and controls. However, plasma
testosterone levels were reduced by approximately 35% in the medium-dose
daidzein group and by 38% in the high-dose daidzein group compared with
controls, without accompanying significant changes in the plasma levels of
either LH or FSH. In contrast, plasma testosterone levels in DES-treated rats
were reduced to almost 22% of the control value, concomitant with reductions
in LH and FSH plasma levels.
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Immunohistochemistry Staining for ER and ERβ
As shown in Figure 3,
immunodetectable ER was weak or very weak in the corpus cavernosum of
control (Figure 3A), daidzein
(Figure 3B–D), or DES
(Figure 3E) groups. There were
no statistically significant differences among any of the experimental,
DES-treated, and control groups in ER staining score
(Figure 4). ERβ staining
was much stronger in the corpus cavernosum (distributed mainly in endothelial
and vascular smooth muscle cells) in rats from the control groups
(Figure 3F); however, it became
largely negative (Figure
3H–J), and its immunostaining score decreased markedly after
medium- or high-dose daidzein or DES treatment
(Figure 4). ERV staining in the
low-dose daidzein group (Figure
3G) was slightly weaker than that of the controls, but the
staining score was not statistically different between the 2 groups
(P > .05).
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Discussion |
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In this study, we used both an apomorphine test and intracavernous pressure determination to evaluate erectile function. Apomorphine is a nonselective dopaminergic receptor agonist. When administrated in low doses (80 µg/kg), it induces a consistent pattern of erections in normal adult male rats (Hsieh et al, 2004). Using this animal model, we observed that erectile responses to apomorphine were inhibited in a dose-dependent manner when daidzein was administered. Apomorphine-induced erections are androgen dependent (Brien et al, 2000). Thus, it is reasonable to believe that the attenuation of apomorphine-elicited erections observed in daidzein-treated rats is at least partly attributed to androgen deficiency. Intracavernous pressure has been extensively used to evaluate erectile function in animal models (Finberg et al, 1993; Pescatori et al, 1993). Reductions in the intracavernous pressure levels are suggestive of erectile dysfunction (Quinlan et al, 1989). In this study, we observed that the intracavernous pressure levels were reduced significantly in animals treated with a relatively large amount of daidzein. It has been reported that testosterone plays important roles in both the central and peripheral neural pathways for the maintenance of erectile capacity, and androgen deprivation results in significantly decreased intracavernous pressure levels in rats (Suzuki et al, 2007). It may be speculated that testosterone deficiency also contributes to the lower intracavernous pressure levels as a result of daidzein treatment.
It is believed that nitric oxide plays a key role in achieving and maintaining penile erection by initiating smooth muscle relaxation (Burnett, 1997). Impaired release of nitric oxide will lead to erectile dysfunction if the mechanism of smooth muscle relaxation mediated by nitric oxide was crimped. It has been reported that estrogen and genistein (another common form of isoflavone) could increase endothelial nitric oxide synthase via an ERβ-dependent mechanism (Gingerich and Krukoff, 2005) and stimulate nitric oxide release from endothelial cells (Yang et al, 2000; Räthel et al, 2005). The expression of ERs (mainly ERβ) in the penis (Mowa et al, 2006) would suggest that estrogen through activation of the ER may play a role in regulation of intracavernosal smooth muscle tone by affecting nitric oxide production. In this study, we observed that ERβ expression was markedly decreased in the corpus cavernosum after treatment with medium- or high-dose daidzein or DES. Thus, it may suggest that the significant decrease in levels of ERβ in the corpora cavernosa might result in nitric oxide reduction and subsequently lead to the lower intracavernous pressure levels and apomorphine-induced erections as observed in this study. Further studies are needed to elucidate the mechanisms involved.
The negative effects of daidzein on erectile function observed in this study are not identical but are qualitatively similar to those seen in the DES group. Given this phenomena, it suggests that the estrogen-like activity of daidzein apparently underlies the mechanism involved in erectile dysfunction caused by daidzein. However, daidzein was observed to result in only limited reduction in testosterone levels and did not affect penile size and weight. Daidzein structurally resembles estradiol and has estrogenic activity but with a binding affinity to the ER 100 to 1000 times less than estradiol (Kaldas and Hughes, 1989). This may explain that daidzein has only a limited impact on plasma testosterone levels and does not lead to any gross changes in the penis.
The estrogenic potency of daidzein may mean that it acts as an endocrine disruptor that could affect the endocrine system and cause reproductive disturbances if exposed in a large amount (West et al, 2005). It has been estimated that the dietary intake of phytoestrogen is ordinarily 25 to 50 mg per day in some Asians populations (Adlercreutz and Mazur, 1997). Unfortunately, dietary assessment is always imprecise because of differences in dietary habit and preferences in food among people. Soybeans and soybean-based food are rich in isoflavones (1 g of soybeans = 1 mg of isoflavones); therefore, it would be possible for a person to receive significant quantities of isoflavones in soybean foods if they were taken in excess. For these reasons, the dose of daidzein used in the present experiment may also be of concern for male reproductive function in humans. It has been reported that long-term administration of the phytoestrogen genistein could inhibit Leydig cell steroidogenesis ex vivo (Svechnikov et al, 2005). Considering that daidzein is similar to genistein in structure and biologic activity (Hwang et al, 2006), it is reasonable to assume that daidzein may also inhibit Leydig cell steroidogenesis and cause plasma testosterone levels to be reduced, as observed in this study.
Taken together, our novel findings of the impact of daidzein exposure during puberty on penile erection provide clear evidence that exposure of relatively large amounts of daidzein in juvenile rats could adversely affect erectile capacity in adulthood; however, penile growth was not affected. Androgen deficiency, as well as reduction in the expression of ERβ in the corpora cavernosa, may at least in part account for the attenuation of erectile function. The evidence suggesting that dietary daidzein may be a factor contributing to erectile dysfunction represents a novel line of epidemiologic thinking. Further experimental, and particularly epidemiologic, studies are required to advance our understanding of the impact of phytoestrogens on erectile function.
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Acknowledgments |
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