1 Introduction

2 Materials and methods

2.1 Animals

Adult male Sprague-Dawley rats (250-300 g) were used and were divided at random into groups of five: the sham operated controlsC, the castrated controlsOx, castrated and treated with TOx+T, castrated and treated with DHTOx+DHT, and castrated and treated with T and MK-434, a 5-reductase inhibitorOx+T+MK434. Animals were sham operated or castrated under intraperitoneal ketamine anesthesia (35 mg/kg). T or DHT treatment included the subcutaneous implantation of Silastic brand silicon tubing containing powdered T or DHT (Dow Corning, Midland, Mich; OD, 3.17 mm; ID, 1.57 mm) immediately after castration for 7 days, which has been shown to induce physiological serum T or DHT levels in adult rats^[4]. In one group of Ttreated rats, MK-434 (Merck Research Laboratories, West Point, Pa.) 1 mg/kg was injected daily in order to inhibit the conversion of T to DHT. Animals were maintained under standard housing conditions with free access to tap water and laboratory chow. Serum T and DHT levels were measured by radioimmunoassay. For measurement of NOS activity, serum T was determined in samples taken after ES or penectomy.

2.2 Measurement of penile reflex

Penile reflex tests were conducted between 08:30 and 13:30 h; the animal was placed in the supine position in a cylinder for 25 min. After 5 min of adaptation, the penile sheath was rolled back with a cotton-tipped wooden applicator to expose the glans^[6]. The occurrences of glans engorgement, cup, and flip were recorded on an event recorder for a period of 20 min from the first occurrence of any erectile response. Glans engorgement was scored when the penis became engorged and swollen; cup involved the flaring out of the tip of the phallus to a circumference equal to or greater than the base of the glans; flip involved a dorsal deflection of 30\+o or more from the resting position^[6,7].

2.3 Measurement of erectile response to electrical stimulation (ES)

Rats were anesthetized with intraperitoneal injection of ketamine (35 mg/kg), and the erectile response was measured as previously described^[1]. Briefly, after surgical exposure of the hypogastric and pelvic nerve, the major pelvic ganglion, and the cavernous nerves, the last-named were stimulated with a glass electric stimulator and a bipolar platinum electrode (stimulus magnitude: 1 to 3 V; frequency: 20 Hz; duration: 5 ms). During stimulation, erections were evaluated by monitoring the intracavernous pressure (ICP). After the ischiocavernous muscle was divided, the silver-white colored tunica albuginea of the penile crus was exposed. A 22-gauge butterfly needle was inserted into the penile crus and connected to the monitor. During the experimental period, the animals were injected 35 mg/kg of ketamine every 45 min to maintain the anesthesia. Systemic blood pressure (SBP) was monitored by inserting a polyethylene tube (PE-50) into the right femoral artery.

2.4 Measurement of NOS activity

To determine whether changes in erectile response to ES under various hormone status correlated with variations in the ability of the penis to synthesize NO, NOS activity was measured in the soluble fraction of the penis. NOS activity was determined indirectly by measuring the nitrite concentration, using Griess reaction^[8]. The half-life of NO in vivo is only a few seconds; most NO is oxidized to nitrite (NO₂^-)/nitrate (NO₃^-), and the concentration of these anions has been used as a quantitative index of NO production. Nitrite concentrations in tissue homogenates were determined in the penis of animals not subjected to ES; this precaution was essential to avoid NOS activation or inhibition induced by the ES itself. The procedure was basically the same as that described before^[4,9]. Briefly, rats were anesthetized with ketamine, and the penile bulb and shaft (excluding skin and glans) were excised. Homogenates were prepared in four volumes of cold solution containing 0.32 mol/L sucrose, 20 mmol/L HEPES (pH 7.2), 0.5 mmol/L EDTA, and 1 mmol/L DTT, using a Polytron homogenizer, and centrifuged at 12 500g at 4 for 15 min. To 200 L of supernatant, 10 L each of 2 mmol/L arginine and 2 mmol/L NADPH were added, and the mixture was incubated  at 37 for 1 h. Following the addition of 200 L Griess reagent (1% sulfanylamide, 0.1% naphthylethylene diamine, and 2.5% H₃PO₄), the mixture was incubated for 10 min at room temperature. Nitrate concentration was measured by comparing with a standard curve, using linear gradient concentrations of sodium nitrate.

2.5 Statistical analysis

3 Results

3.1 Serum T and DHT levels

Figure 1 gave the serum T levels in different groups. It can be seen that after castration, the serum T was markedly decreased. It was restored by T but not by DHT supplementation. The combined use of T and MK-434 restore the T level to a much lower value.

Figure 1. Serum testosterone levels. (1.760.46) ng/mL in groupC, <0.01 ng/mL in groupOx, (1.780.37) ng/mL in group Ox+T, (1.290.37) ng/mL in group Ox+T+MK434, and<0.01 ng/mL in group Ox+DHT.

In Figure 2, the serum DHT levels are shown. It was decreased after castration and was restored by T supplementation, but not by the combined use of T and MK-434. DHT supplementation raised the serum DHT to almost double the control level. The DHT levels in groupsOx+T+MK434andOxwere more or less similar, signifying the effective inhibition of 5-reductase activity by MK-434.

Figure 2. Serum DHT levels. (64.714.6) g/mL in groupC, (15.256.03) g/mL in group Ox, (57.821.4) g/mL in group Ox+T, (19.76.1) g/mL in group Ox+T+MK434, and (112.820.8) g/mL in group Ox+DHT.

3.2 Effect of castration and androgen replacement on penile reflex

The data were expressed as percentage of the level in the control group in consideration.  

3.2.1 Glans engorgement (Figure 3A)

Castration did not induce a significant decrease in the number of erections as compared with group C (66.7%46.5%, P=0.54). Administration of T increased the number of erections to significantly above the group C level  (222%152.1%, P=0.20); a similar increase was obtained with DHT administration.

The combined use of T and MK-434 caused a statistically significant reduction in the number of erections as compared with the T-treated group (30.0%20.8%, P<0.05).

3.2.2 Cup (Figure 3B)

Castration reduced the number of cups to 9.1%8.3% (P=0.021) of groupC, but with T or DHT supplementation, the number was restored to 87.9%50.0% (P=0.76) or 109.1%89.3% (P=0.86), respectively. The combined use of T and MK-434 caused a statistically significant reduction in the number of cups as compared with groupC(24.2%17.3%, P=0.048).

3.2.3 Flip (Figure 3C)

Castration reduced the number of flips to 7.1%9.7% (P=0.01) of groupC. It was restored to 67.9%67.3% (P=0.21) with T supple mentation. The administration of DHT, however, did not cause a similar increase in the number of flips (25.0%39.1%, P=0.04). The combined use of T and MK-434 caused a statistically significant reduction in the number of flips as compared with the T-treated group (25.0%20.4%, P=0.02).

Figure 3. Effects of castration and androgen replacement on reflex erections: (A) glansengorgement, (B) cups, (C) flips.

3.3 Effect of castration and androgen replacement on the erectile response to ES

Castration induced a considerable decrease in erectile response to 59.9%11.6% (P<0.001) of groupC. Administration of T to castrated rats restored the erectile response to a level similar to group C. The administration of DHT led to a similar normalization of penile response to ES. When using variance analysis to compare the castrated group with groupC, the 40 % reduction induced by castration in response to ES was significant (P<0.001). Erectile responses to ES in T or DHT treated rats were 100.9%27.5% (P=0.942) and 108.5%7.2% (P=0.125) of groupC, respectively. Erectile response of the androgen supplemented groups were not significantly different from those of group C. The combined use of T and MK-434 caused a statistically significant reduction in the erectile response to ES (70.5%20.2%, P=0.012). (Figure 4)

Figure 4. Effects of castration and androgen replacement on penile erectile response to ES. Intracavernous pressure (ICP); systemic blood pressure (SBP).

3.4 Effect of castration and androgen replacement on penile NOS levels

In all the five groups of animals, the pattern of changes in the NOS activity was similar to that in the erectile response to ES. Castration reduced penile NOS activity to 32.4%12.7% (P<0.001) of groupC; the activity was restored by T or DHT supplementation to levels of 90.2%16.0% and 97.1%13.8%, respectively, which were not significantly different from the group C (P=0.371, P=0.762, respectively). In theOx+T+MK434 group, the enzyme activity was 45.0%15.0% (P<0.001) of group C. (Figure 5) 

4 Discussion

Reflex penile erections in the rat have been previously described, but the neurophysiology remains poorly elucidated. There has been no convincing evidence that reflex erectile responses are induced through sinusoidal dilation via the autonomic cavernous nerve rather than through contraction of bulbocavernous and ischiocavernous muscles via the somatic pudendal nerve. Giuliano et al^[10], however, reported that in anesthetised rats, striated muscle paralysis did not abolish the increase in intracavernous pressure induced by electrical stimulation of the dorsal nerve of the penis. By measuring the increased intracavernous pressure with telemetric device and perineal muscle activity during reflex erections, Bernabe et al^[11] demonstrated the involvement of the corpus cavernosum in reflex responses. These data help to define the participation of the spinal reflex arc, corpus cavernosum and perineal muscles in  reflex penile erections. In this study, penile reflex erection in rats was shown, on the whole, to be related to serum T levels, even though the number of glans engorgements was not. The number of cups and flips was significantly reduced by castration, but was restored to the control level by T supplementation.

It has been suggested that androgen levels influence corpus cavernous smooth muscle relaxation via alteration in NOS activity^[4,8,12,13]. In this study, the erectile response to ES and the NOS activity in penile tissue were related to serum T level, and this is an ample evidence of the direct effect of androgen on corpus cavernous smooth muscle relaxation.

Our results showed that in castrated rate, the administration of T restored the number of reflex erections, and the erectile response to ES and NOS activity in the corpus cavernosum, while the administration of T together with MK-434, a 5-reductase inhibitor, no longer restored these responses. It may imply that T has to be converted to DHT to exert its influence. It is well known that DHT has a higher affinity than T to the androgen receptor^[14]. However, the results of flip experiment appear to be inconsistent with other results: the number of flips was lower in the DHT-treated group than that in the T-treated group. The relative role of T and DHT in flip may require further investigation.

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