Tomi Streng, Department of Clinical and Experimental Pharmacology, Lund University Hospital, SE-221 85 Lund, Sweden. e-mail: firstname.lastname@example.org
To explore the effect of different degrees of oestrogenization on male voiding, by treating adult castrated and 5α-dihydrotestosterone (DHT)-maintained male mice with different doses of oestrogens, as exposure of male mice to excessive amounts of oestrogens can cause bladder outlet obstruction (BOO); in addition, male mice lacking oestrogen receptor (ER)α (ERKO) or ERβ (BERKO) were studied to assess the importance of ER subtypes.
MATERIALS AND METHODS
Castrated, DHT-maintained adult mice were treated with 17β-oestradiol (E2; 50 and 250 µg/kg) or oestrone (E1; 5, 50 and 500 µg/kg) daily for 10 days. Control mice were treated only with the vehicle. BERKO and ERKO mice, and their wild-type littermates used as their controls, remained untreated. Under anaesthesia, the bladder and distal urethra were exposed to record simultaneously the bladder pressure and urinary flow rate from the distal urethra.
E2-treated mice showed obstructive voiding, seen as increased bladder pressure, decreased average flow rate and prolonged micturition time. This was also evident when a high dose (500 µg/kg) of E1 was used. After treatment with a dose of 50 µg/kg, the urodynamic variables were similar to those in the control mice. Surprisingly, after treatment with a low dose (5 µg/kg) all urodynamic variables improved. There was a minor increase in the bladder pressure in BERKO mice; ERKO mice had a significantly lower urinary flow rate.
High doses of oestrogens caused BOO in castrated, DHT-maintained male mice. A small dose of E1 had a positive effect on voiding, suggesting that oestrogens are needed for normal male voiding. Reduced urinary flow rates in ERKO mice suggest that oestrogen effects on voiding are mediated at least partly via ERα.
Adult male mice chronically treated with oestrogens develop BOO with urinary retention, an enlarged bladder with occasional bladder stones and hydronephrosis [1,2]. The same is true for developmentally oestrogenized male mice [3,4]. They have lower voided urine volumes and a lower ratio of urinary flow rate to bladder pressure, both consistent with BOO. Oestrogen-related BOO may at least partly be due to failure of the rhabdosphincter to relax . According to these findings, oestrogens would have an exclusively negative effect on voiding in male animals. However, the dose-response relations for the effects of oestrogens have, to the best of our knowledge, not been studied. Further, the altered androgen concentrations in oestrogen-treated uncastrated males may have hampered the detection of the role of oestrogens.
Thus the aim of the present study was to investigate the dose-response relationship of two oestrogens (17β-oestradiol, E2; and oestrone, E1) on voiding in adult male mice. E2 is the most potent oestrogen in the mammalian organism. E1 was chosen because it is the main circulating oestrogen in males. The androgen concentration was maintained after castration using 5α-dihydrotestosterone (DHT) treatment. This is important, because androgens have been shown to influence bladder activity [6–9]. Further, mice lacking oestrogen receptor (ER)α (ERKO) or ERβ (BERKO) were studied to reveal the significance of both for male voiding.
MATERIALS AND METHODS
Urodynamic variables from 60 male adult mice (C57Bl/Bom strain) were recorded with the mice under anaesthesia. In the first part of the study, the effects of E2, in the second part E1 and in the third the role of ER-α and -β were assessed. The mice were maintained under standard laboratory conditions in a 12 : 12 h light/dark cycle, with free access to soy-free food pellets (SDS, Witham, Essex, UK) and tap water. The Animal Care Organization and University Ethical Committee of Turku University approved the study protocol. The mice were handled in accordance with the institutional animal-care policies of the University of Turku.
Under anaesthesia (Dormicum®, 5 mg/mL; Roche Oy, Espoo, Finland and Hypnorm®, Janssen Pharmaceutica, Beerse, Belgium) adult male mice were castrated and treated with DHT implants s.c. (21-day release pellets, dose 1.5 mg/day; Innovative Research of America, Inc. Sarasota, FL, USA) to sustain a basic androgen level. After a 7-day recovery the treatments began. The mice were weighed just before the treatments and afterwards once a week.
For the E2 study, 27 mice (mean age 209 days, sd 8.8) were used; they were divided into three groups and treated s.c. with E2 (Sigma Chemical Co. St. Luis. MO, USA; 50 or 250 µg/kg in rape-seed oil; Kultasula, Raisio Yhtymä, Raisio, Finland) for 10 days. Control mice were treated with rape-seed oil injections only. Mice were treated for 10 days because in a preliminary 5-day study, the groups did not differ from each other.
For the E1 study, 33 mice (mean age 422 days, sd 39.7) were used, divided into four groups and treated s.c. with E1 (Sigma; 50 or 500 µg/kg in rape-seed oil for 10 days). Control mice were treated with rape-seed oil injections only.
For the BERKO study, 35 mice were used (mean age 144 days, sd 36.3, 16 mice) and their wild-type (WT) littermates were used as controls (144 days, sd 28.6, 19 mice). For the ERKO study, 22 mice were used (mean age 146 days, sd 26.8, 10 mice) with and their WT controls (150 days, sd 11.2, 12 mice). No treatments were given in either group.
For the urodynamic studies the mice were anaesthetized with chloral hydrate i.p. (0.09 g/kg, Sigma) as a basic anaesthetic, and i.v. injection with urethane (0.16 g/kg, Sigma) was used to maintain anaesthesia for urodynamic measurements. The body temperature was kept constant at 36–38 °C by a thermostatically controlled animal blanket (Harvard Animal Blanket Control Unit, type 50–7061, Harvard Apparatus Limited, Edenbridge, UK), and if needed, with a heating lamp. The bladder and the distal part of the urethra were exposed via a midline incision of the lower abdomen. Tissues were kept moist during measurements with warm (37 °C) saline (0.9% NaCl).
For bladder pressure measurements, a 20 G i.v. cannula was inserted through the bladder apex into the lumen . The cannula was connected to an infusion pump (SP 100i Syringe Pump, World Precision Instruments, Inc., Sarasota, USA) and to a pressure transducer (Statham P23XL, Hato Ray, Puerto Rico). The pressure transducers were connected to an amplifier (Model 7P122B, Grass Instruments, Quincy, MA, USA). The whole system was filled with warm (37 °C) saline and continuous infusion of it into the bladder was started after an equilibration time (10 min). Micturition was evoked by the infusion of saline. Measurements were made at an infusion rate of 0.08 mL/min. An ultrasonic flow probe (Probe ♯ 1.5 RB 208, Transonic Systems, Inc., Ithaca, NY, USA) surrounding the distal urethra, and connected to a flow meter (T106, Transonic Systems) was used to measure the urine flow rate . The pressure and flow meter signals were transferred to the Biopac-system (Biopac Systems Inc., Santa Barbara, CA, USA) and connected to a personal computer. Continuous recordings were obtained using Acq Knowledge 3.5.3 software (Biopac). The sampling rate was 400 Hz by the computer software, but the flow signals were already filtered (100 Hz) by the flowmeter.
Three representative voids from each mouse were chosen for further analysis. Maximum bladder pressures were measured during the first (before the bladder pressure oscillations started), second and third phase of micturition contraction. During the second phase, intraluminal pressure high-frequency oscillations (IPHFOs) of bladder pressure are associated the urine flow. In addition, the average bladder pressure was measured from all the IPHFOs during the second phase. The maximum flow rate was measured from the highest urinary flow rate peak and the mean flow rate from all flow peaks during the second phase of micturition. Further, the duration of micturition and the volume of the residual urine were measured as follow. First the bladder was emptied by aspiration with a syringe, during which there was no infusion of saline into the bladder. After emptying the bladder the infusion (0.08 mL/min) was started. During micturition, the voided volumes were collected into tared test tubes, and these volumes subtracted from the infused volumes to get the residual urine.
The normal distribution of the data was assessed using the Shapiro-Wilk W normality test and Levene's test for variances. If they showed significant differences (P < 0.05) between the treatment groups, a Kruskall-Wallis test was used, and the Mann–Whitney U-test was used as a posthoc test. Otherwise, a one-way anova was used to analyse the data between the groups, and the Tukey honest significant difference test used posthoc.
Figure 1 illustrates simultaneous recordings of bladder pressure (A) and urinary flow rate (B) during a micturition contraction in a control male mouse. As in rats , the voiding contractions may be divided into four phases (Fig. 1). The IPHFOs during phase 2 are conspicuous. Neither treatment with E2 or E1 caused any qualitative changes in recordings (Figs 2 and 3). The four phases are recognisable also in the oestrogen-treated mice.
In the first phase of bladder pressure, the maximum bladder pressure was significantly greater after the E2 250 µg/kg dose than in the control mice (Fig. 2; Table 1). The maximum bladder pressure in the E1 5 µg/kg group was not significantly different from that of the control mice, but it was the lowest of the E1 treatment groups (Fig. 3). The maximum pressure was significantly greater in the 500 µg/kg group than in all other groups (Table 2).
Table 1. Changes in urodynamic variables of mice treated with E2 (50 and 250 µg/kg; 10-day treatment; nine mice in each group)
Mean (sd) variable
P, control vs E2; 50, 250
P, E2 50 vs 250
Max BP, maximum bladder pressure, mmHg; Mean BP, mean bladder pressure, mmHg; MF, maximum flow rate, mL/min; AF, average flow rate, mL/min; MT, micturition time, s; RU, residual urine, mL. P calculated by anova, unless *Mann–Whitney U-test.
In the second phase of bladder pressure, the maximum bladder pressure during the IPHFOs was significantly greater with E2 (250 µg/kg) than in control mice. In addition, the mean bladder pressure increased after E2 treatment. The effect on bladder pressures did not differ between the E2 doses (Table 1). The maximum bladder pressure was significantly greater in the E1 500 µg/kg group than in all the other groups. The mean bladder pressure also was greater than in the 5 µg/kg treated mice (Table 2).
In the third phase of bladder pressure the maximum bladder pressure was significantly greater in E2 (250 µg/kg) treated mice than in control mice (Table 1). In the E1-treated mice the maximum bladder pressure was significantly greater with 500 µg/kg than 5 µg/kg (Table 2).
The urine flowed during the second phase of micturition; the maximum flow rate did not differ between the control and the E2-treated mice but the mean urine flow rate, which covers all flow peaks during one micturition cycle, was significantly lower in all E2-treated mice (Table 1). In the E1-treated mice the 5 µg/kg dose gave the highest maximum and mean urinary flow rates. Three of seven mice had at least as high a value as the highest of the controls, both in maximum and mean flow rates. The mean flow rate was significantly lower with the two higher concentrations than in the lowest (Table 2).
The duration of micturition was prolonged in both groups of E2-treated mice compared to the control mice. In addition, the micturition time was significantly longer in the 250 µg/kg than in the 50 µg/kg group (Table 1). In the E1-treated mice the micturition time was not significantly different from that of the control group (Table 2). However, the micturition time was significantly longer in the 500 µg/kg than in the 5 µg/kg treated mice (Table 2).
The amount of residual urine was not statistically different between the treatments and control mice. There was a slight (0.03 mL) but significant increase in the E1-treated mice (500 µg/kg) vs the 5 µg/kg-treated mice (Tables 1 and 2), but no change in the bladder capacity when compared with the control mice (data not shown).
ERKO mice had a lower maximum (sd), at 7.9 (2.22) vs 14.2 (5.25) mL/min (P < 0.001), and mean urinary flow rate, at 1.0 (0.31) vs 1.5 (0.34) mL/min (P = 0.006) than their littermates. The other variables were no different from those of the WT littermates. There was a greater mean bladder pressure, at 20.3 (4.04) vs 17.2 (3.84) mmHg (P = 0.02), in BERKO mice but otherwise the urodynamic variables were not significantly different in BERKO mice than their WT littermates.
There are several studies showing that exposure to excessive amounts of oestrogens can cause BOO. The present results suggest that the effects of oestrogens may be dual and dose-dependent. First, we confirmed the obstructive effect of high doses of E2 in adult castrated male mice maintained with DHT. E2 treatment at 50 or 250 µg/kg increased the bladder pressure during all phases of the micturition cycle. Even though the maximum flow rate was unaffected, the mean flow rate was lower after E2 treatments. The prolonged micturition time further confirmed the presence of BOO. There were no statistically significant differences between the dose groups except in the micturition time, which was considerably longer at the higher dose. Second, there was a surprising finding in the E1-treated mice; there were obstructive effects at the highest dose (500 µg/kg) as expected, when E2 is assumed to have 10 times the potency of E1. At 50 µg/kg, E1 had no statistically significant effects on the urodynamic variables when compared with control mice. The most straightforward explanation would be that the oestrogenicity of the dose and/or the conversion of E1 to E2 in the organism is too low to cause obstructive voiding. However, when the mice were treated with the low dose of E1 (5 µg/kg) the variables measured showed no sign of obstructive voiding. Third, the ERKO mice had lower urinary flow rates and the BERKO mice had a higher mean bladder pressure than their WT littermates. Together, these findings suggest that oestrogens may be needed for normal voiding of the male mouse.
The mechanisms and the sites of oestrogen action in the LUT have not been established. The present findings in ERKO mice suggest not only that oestrogen is needed for to maintain a normal flow rate but also that the increase in the urinary flow rate after treatment with the lowest dose of E1 could be mediated through ERα. There are only a few ERα positive cells in the LUT of the male mouse , which would make a local effect in the tract through ERα unlikely. However, ERα may be involved in neuronal control of the LUT. ERα positive cells have been found in autonomic and spinal ganglia, and various structures of the CNS innervating the LUT . Oestrogen treatment has been shown to induce a moderate increase in noradrenaline sensitivity  that would promote urine storage by increasing urethral resistance and depressing detrusor contractions . Interestingly, Matsubara et al. showed that selective β3-receptor agonists have an enhanced relaxing effect on the detrusor of female rats with low, but not normal, oestrogen levels. It is known that low oestrogen levels are associated with increased voiding frequency , and it was shown that a short (4-day) oestrogen treatment markedly increased the response to stimulation of the muscarinic receptors in the mouse bladder . However, in the rabbit, opposite effects on muscarinic receptor density with longer oestrogen treatment was reported . Based on available knowledge it is difficult to explain how these effects may lead to the present finding that a low dose of E1 caused no obstructive voiding. On the contrary, there was a trend to a lower bladder pressure and greater flow rate in these castrated, DHT-maintained male mice. In the study of Schroder et al., a lack of ERα and/or ERβ had little effect on in vitro contractility of the bladder or on continuous cystometry of female mice.
As the BERKO mice had a higher mean bladder pressure (during the second phase of micturition), and the small dose of E1 decreased bladder pressure, the possibility that ERβ may also be involved in maintaining normal voiding cannot be excluded. The ERβ is the predominant form of ER in the bladder . ERβ has been detected in bladder, urethral epithelium and detrusor muscle . Prostatic ganglia , and the rhabdosphincter also have ERβ. Even though the role of ERβ is still unknown its presence in several LUT structures could make direct effects of oestrogens possible. Particularly the co-expression of ERβ and androgen receptor at many sites and in many cell types in the LUT makes the interaction of oestrogens and androgens an intriguing possibility . The castrated mice were maintained with DHT, which is converted to 3α and 3β, 17β-andostadiols. 3β–andostandiol shows higher affinity for ERβ than ERα; it also induces oestrogenic responses and may mask some ERβ-mediated oestrogenic responses in the present DHT-maintained, castrated mice. Further studies are needed to reveal the possible role of ERβ in the LUT.
In conclusion, high-dose oestrogen treatment may cause BOO in adult male mice but a low dose of E1 may improve LUT function, suggesting that low concentrations of oestrogens could be needed for effective voiding function. The subtype mediating the oestrogen effects on LUT function seems to be ERα, whereas the role of ERβ is unclear.
We thank Professor Pirkko Härkönen for providing the ERKO and Professor Jan-Åke Gustafsson for the BERKO mice. Mr Jarmo Immonen assisted with urodynamic measurements. This study was supported by National Technology Agency of Finland (TEKES) (grant number: 40244/99).
CONFLICT OF INTEREST
None declared. Source of funding: National Technology Agency of Finland (TEKES).