Introduction

Research in male sexual dysfunction has predominantly focused on erectile dysfunction. However, premature ejaculation is the most common male sexual disorder, estimated to affect up to 30% of men.¹ Despite its prevalence and adverse impact on patient quality of life, relatively little attention has been focused on investigating the causes of premature ejaculation or developing therapeutic strategies. Patient demand for effective treatment will inevitably spark development of new therapeutic agents, with particular interest in serotonergic drugs.

Behavioral studies in animals suggest that the serotonergic system is a primary regulator of the ejaculatory reflex.^{2, 3} Recently, the selective serotonin reuptake inhibitors (SSRIs) have emerged as an effective new treatment modality for patients with premature ejaculation.^{4, 5} The clinical efficacy of SSRIs also provides additional evidence that serotonin (5-hydroxytryptamine; 5-HT) plays an important role in ejaculation.

It is of critical importance to develop a versatile in vivo model to assess therapeutic efficacy of drugs used in the treatment of premature ejaculation. Previous efforts have been made to develop an in vivo model to measure the contractile response in the seminal vesicle or vas deferens to electrical nerve stimulation in order to evaluate therapeutic efficacy of drugs for premature ejaculation.^{6, 7} Aside from the differences between these models in terms of target organs and stimulated nerve, there were qualitative differences in the findings of these two reports.

This report presents modifications of these earlier attempts to offer an in vivo model in which the intraluminal pressures of the seminal vesicle and vas deferens can be measured in a single animal. Furthermore, we investigated the efficacy of several drugs used in the treatment of premature ejaculation.

Materials and methods

Animals

All studies were approved by the Institutional Animal Care and Use Committee. Male Sprague–Dawley rats (300–350 g) were equally divided into eight groups (n=10 per group) based on experimental agents: serotonin, clomipramine, fluoxetine, sertraline, paroxetine, prazosin, terazosin, and tamsulosin. Rats were anesthetized with intraperitoneal injection of ketamine (120 mg/kg). Anesthetized rats were secured in the supine position, and a 2-cm midline neck incision was made to access the carotid artery and expose the external jugular vein. To monitor systemic blood pressure continuously, a 24-guage angiocatheter was introduced into the carotid artery. Intravenous drug administrations were accomplished via the external jugular vein.

Hypogastric nerve stimulation (HNS)

A 3-cm lower abdominal midline incision was made to visualize the bladder, prostate, seminal vesicle, and rectum after displacing the intestine rostrally by packing the abdominal cavity with gauze. Using a surgical microscope, the hypogastric nerves were identified. The hypogastric nerves originate bilaterally from the inferior mesenteric ganglion and travel toward the ipsilateral major pelvic ganglion and can be identified in the area adjacent to the ipsilateral ureters. Using microsurgical technique, both hypogastric nerves were delicately dissected away from the surrounding connective tissue from their origin at the inferior mesenteric ganglion to the area adjacent to the seminal vesicle. Then, the nerve was transected proximally just below the inferior mesenteric ganglion (Figure 1).

Figure 1.

Both hypogastric nerves were transected proximally just below the inferior mesenteric ganglion (arrows indicated).

Full figure and legend (74K)

Each hypogastric nerve was mounted on a bipolar silver electrode (Grass SD9, Grass Instrument, Quincy, Massachusetts, USA) and stimulated by a 10 s train of square waves with 6 V pulse amplitude (normal polarity) and 1 ms pulse duration at 1 h after transection of the proximal hypogastric nerve. The stimulation frequency (submaximal frequency) was 40 Hz for the vas deferens and 80 Hz for the seminal vesicle, which caused submaximal contractile responses of each organ through the preliminary experiments.

Pressure measurements in the seminal vesicle and vas deferens

One testis was passed into the abdominal cavity, and the gubernaculum was transected. Using a surgical microscope, a transverse incision was made in the wall of the vas deferens about 2 cm from the epididymis. After exposing the lumen of the vas deferens, a polyethylene tube (PE-10) filled with normal saline was inserted into the lumen of the vas toward the prostate gland and secured.

After unraveling the tail of the contralateral seminal vesicle, an obliquely cut polyethylene tube (PE-60) filled with normal saline was delicately inserted into the main lumen of the seminal vesicle and tightly secured to prevent leakage of seminal fluid. The tubes for monitoring blood pressure and intraluminal pressures of the vas deferens and contralateral seminal vesicle were connected to pressure transducers and a polygraph (TA6000, Gould Instrument Systems, Valley View, Ohio, USA). The selection of right or left organs for dissection was randomly determined. The intraluminal pressure of the vas deferens, the contralateral seminal vesicle and systemic arterial pressure were continuously recorded throughout the experiments.

In vivo study protocol

The control responses to HNS at submaximal frequency were determined in each animal. Upon return to baseline values, animals received intravenous administration of the drug in saline. Serotonergic drugs (serotonin, clomipramine, fluoxetine, sertraline, and paroxetine) and -adrenergic blockers (prazosin, terazosin and tamsulosin) were given intravenously by cumulative injection in the order of 0.01, 0.1, 1, 5, 10 mg/kg and 0.0001, 0.001, 0.01, 0.1, 1 mg/kg, respectively. HNS was repeated sequentially 20 min after the administration of each drug, and the intraluminal pressures of the vas deferens and contralateral seminal vesicle were measured separately.

Data analysis

All data are reported as means.d. Relative potencies were determined based on the extrapolated concentrations at which 50% inhibition of intraluminal pressure responses (EC₅₀) was observed. A repeated measures ('mixed model') two-way (drug and concentration) analysis of variance (ANOVA) was conducted on the data. A priori comparisons were performed using Bonferroni's method. Means were considered to be significantly different for P-values less than or equal to 0.05. All statistical analyses were performed using GraphPad PRISM version 4.0 software.

Results

HNS caused significant increases in intraluminal pressure in the seminal vesicle and vas deferens. Prior to drug administration, the peak intraluminal pressures of the seminal vesicle and vas deferens subsequent to HNS were 746.8 and 565.6 cm H₂O, respectively. Repeated nerve stimulation, each preceded by a 20-min rest interval, produced reproducible intraluminal pressure recordings in each organ. Also, baseline values of intraluminal pressure did not vary significantly after intravenous injection of the agents at the doses administered in this study.

All serotonergic drugs investigated in this study elicited a concentration-dependent decrease of the HNS-induced intraluminal pressure elevations in the seminal vesicle (Figure 2a). In the vas deferens, 5-HT and clomipramine demonstrated significant concentration-dependent effects, while the SSRIs did not have a significant effect (Figure 2b). The administration of 10 mg/kg clomipramine proved to be fatal, and the corresponding data points were eliminated from analysis. All -blockers administered in the course of this study elicited concentration-dependent decreases in the HNS-induced elevations of the seminal vesicle (Figure 3a) and vasal pressures (Figure 3b). The administration of serotonergic drugs and -adrenergic inhibitors at the concentrations included in this study did not significantly alter the systemic blood pressure in the animals.

Figure 2.

Effects of serotonergic drugs on intraluminal pressure of the seminal vesicle (a) and vasa deferentia (b). The seminal vesicle pressure: F_4,120=8.90, P<0.0001; vasal pressure: F_4,120=6.92, P=0.0005.

Full figure and legend (31K)

Figure 3.

Effects of -adrenergic blockers on intraluminal pressure of the seminal vesicle (a) and vasa deferentia (b). The seminal vesicle pressure: F_2,90=30.69, P<0.0001; vasal pressure: F_2,90=1.38, P=0.2780.

Full figure and legend (28K)

The relative potencies of the serotonergic drugs in the seminal vesicle and vas deferens were determined by extrapolating the EC50's. The EC50's for clomipramine, 5-HT, fluoxetine, sertraline, and paroxetine in the seminal vesicle were 0.16, 7.1, >10, >100, and >100 mg/kg, respectively. The corresponding values in the vas deferens were 5.2, 2.5, >100, >100, and >100 mg/kg, respectively. The sympatholytic drugs tested (prazosin, terazosin, and tamsulosin) proved to be significantly more potent in the seminal vesicle than the vas deferens. At 10 mg/kg, sympatholytic drugs virtually abolished any HNS-induced response in the seminal vesicle, while approximately 40% of the response was still observed in the vas deferens.

Discussion

Serotonin appears to be the most important neurotransmitter in the control of the ejaculatory reflex. The most convincing evidence of serotonin action and its influence on ejaculatory function comes from the observed clinical efficacy of SSRIs in premature ejaculation.^{4, 5} The involvement of central serotonergic neurotransmission in ejaculation has been investigated in animal studies. The drug stimuli of the SSRIs, fluoxetine, and paroxetine, most closely resemble 5-HT2C receptor activation.⁸ The activation of both 5-HT2A and 5-HT2C receptors by 2,5-dimethoxy-4-iodophenyl-2-aminopropane increases ejaculatory latency,⁹ while the selective 5-HT2A receptor agonist 2,5-dimethoxy-4-methylamphetamine does not exhibit this effect.¹⁰ On the other hand, selective activation of 5-HT1A receptors by 8-hydroxy-2-(di-n-propylaminotetralin) shortens the ejaculatory latency time and reduces the number of intromissions preceding ejaculation in animals.¹⁰ Based on these animal studies, Waldinger et al¹¹ hypothesized that premature ejaculation was caused by a disruption in the functional balance of 5-HT receptor subtypes: increased sensitivity of 5-HT1A receptors with decreased sensitivity of 5-HT2C receptors.

Development of new serotonergic drugs seems inevitable in light of the high incidence and the need for effective treatment of premature ejaculation. Thus, the development of an in vivo experimental model for assessing the efficacy of these new drugs is a pressing matter. Hsieh et al⁶ proposed a rat model involving electrical stimulation of the lesser splanchnic nerve to induce changes in the intraluminal pressure of the seminal vesicle. They measured the inhibition of electrical stimulation-induced seminal vesicle pressure increases by several agents (prazosin, 5-HT, clomipramine, fluoxetine, imipramine, and indatraline). They observed concentration-dependent effects with prazosin and all serotonergic agents, except imipramine and indatraline. Of these inhibitory agents, they reported the highest observed efficacy with fluoxetine followed by prazosin, 5-HT, and clomipramine. On the other hand, Kim et al⁷ implemented a somewhat different model that contests the findings of the above study. They investigated the effects of serotonergic drugs (clomipramine, sertraline, paroxetine, and fluoxetine) on the vasal pressure induced by electrical stimulation of the hypogastric nerve. All serotonergic drugs caused concentration-dependent inhibition of intraluminal pressure elevation in the vas deferens. And, clomipramine showed the strongest inhibitory effect followed by sertraline, paroxetine, and fluoxetine, which exhibited the greatest efficacy in the above-mentioned study.⁷

Aside from the technical differences between these models in terms of target organs (seminal vesicle vs vas deferens) and stimulated nerve (lesser splanchnic nerve vs hypogastric nerve), there were qualitative differences in the findings of these two reports. To settle this matter, we modified these previous models to devise a new in vivo experimental model in which the intraluminal pressure responses of both the seminal vesicle and vas deferens can be measured in a single animal. We chose to stimulate the hypogastric nerve because it might be more specific in action or distribution than the lesser splanchnic nerve to induce contraction of the seminal tract by electrical stimulation, as the former is a postganglionic branch from the inferior mesenteric ganglion while the latter is located cranial to the ganglion.¹² In the human, Learmonth¹³ demonstrated that electrical stimulation of either the presacral nerve (superior hypogastric ganglion) or hypogastric nerves produces contraction of the bladder neck, seminal vesicles, vasa deferentia, and ejaculatory ducts.

The basic hypothesis of our study is that the drugs for the treatment of premature ejaculation produce their effects by inhibiting intraluminal pressure elevations of the seminal tract. All serotonergic agents significantly inhibited the increase in the seminal vesicle pressure induced by HNS. On the other hand, the vasal pressure responses were not significantly inhibited by the SSRIs (sertraline, fluoxetine, and paroxetine), which have been demonstrated to have therapeutic efficacies for premature ejaculation.^{4, 5} With 50–70% of the ejaculatory volume accounted for by the seminal vesicle, it appears that this organ is a greater contributor than the vasa deferentia in ejaculation.¹⁴ Thus, it is worth noting that all serotonergic drugs demonstrated the significant inhibitory effects on the contractile responses of the seminal vesicle, whereas minimal (sertraline) to no effects (fluoxetine and paroxetine) with SSRI administration were observed in the vasal pressure responses.

Comparison of the therapeutic efficacies of the serotonergic drugs is currently still not feasible due to a lack of knowledge about the exact nature of the relationship between the seminal vesicle pressure and emission or ejaculation. In fact, one of the fundamental shortcomings of this and other animal models investigating contractile responses of the seminal tract is that these models effect electrical stimulation-induced emission rather than true ejaculation. The minimum concentration or pressure range, at which a drug demonstrates an effect, whether by causing ejaculatory delay or outright suppression of the ejaculatory reflex, has not been determined. Since we cannot yet explain exactly the pathophysiology of premature ejaculation, we cannot yet correlate the inhibition of elevated intraluminal pressures with the clinical target effect of ejaculatory delay.

One of the common strategies of studies thus far (including this study) is that investigators wait for only a short period of time (10–30 min) after drug administration to perform the nerve stimulation. Thus, these studies are limited to observing the acute effects of the drugs. This is a bit counterintuitive when considering the well-documented multiple day lag time before the observation of ejaculatory delay in premature ejaculation patients treated with SSRIs. In fact, the mild delaying effect in humans of fluvoxamine on ejaculation relative to paroxetine was demonstrated in a placebo-controlled, male rat study using a chronic administration treatment model.^{4, 15} However, this difference in the effects of these two agents has not been observed in an acute treatment model.¹⁶ For this reason, we are currently in the process of revisiting this model in rats that are fed serotonergic drugs daily for 1 week or more. Such a modification may make this model more suitable for use as a system in which therapeutic potentials of drugs for the treatment of premature ejaculation can be assessed.

In the present study, all -blockers tested (prazosin, terazosin, and tamsulosin) significantly inhibited the increases in the seminal vesicle and vasal pressures. These observations are not surprising because emission and bladder neck closure are primarily controlled by sympathetic nerves acting via -adrenergic receptors.¹⁷ The greater effects of these agents in the seminal vesicle than in the vasa deferentia may be related to the predominance of 1-adrenoceptors in the rat seminal vesicle.¹⁸ These results are consistent with those of Hsieh et al,¹⁹ who reported that sympatholytic drugs effectively attenuated contractile responses in the seminal vesicle induced by electrical nerve stimulation of lesser splanchnic nerve in rats. Although the -blockers included in this study served as a positive, internal control to confirm the viability of this modified rat model, Cavallini demonstrated the therapeutic efficacy of 1-adrenergic blockers (alphuzosin and terazosin) for premature ejaculation in a double-blind, placebo-controlled, crossover clinical trial.²⁰ However, to explain the fact that the in vivo effect of -blockers in these experiments far exceeds their clinical efficacy, experimental modifications are needed to evaluate the expulsion phase of ejaculation. Development of such in vivo animal models underscore the need for more focused research in the elucidation of the physiology of ejaculation and highlight the importance of a robust model for the investigation of therapeutic efficacy for pharmacological agents in the treatment of premature ejaculation.

Conclusions

This model presents a viable approach for the assessment of serotonergic drug action in the seminal vesicle and vas deferens, and illustrates the importance of the hypogastric nerve for the stimulation of smooth muscle contractions in the seminal tract. In light of the known clinical efficacy of SSRIs in the treatment of premature ejaculation, the differential effect of these drugs observed in this study at the seminal vesicle and vas deferens suggest that the seminal vesicle might be the most important target organ to monitor in the ejaculatory response. Consequently, the measurement of HNS-induced seminal vesicle pressure response may be useful for the evaluation of drugs for the treatment of premature ejaculation.

References

1. Derogatis LR. Etiologic factors in premature ejaculation. Med Aspects Hum Sexuality 1980; 14: 1168–1176.
2. Ahlenius S, Larsson K, Svensson L. Further evidence for an inhibitory role of central 5-HT in male rat sexual behavior. Psychopharmacology (Berl) 1980; 68: 217–220. | PubMed |
3. McIntosh TK, Barfied RJ. Brain monoaminergic control of male reproduction behavior. I. Serotonin and the post-ejaculatory refractory period. Behav Brain Res 1984; 12: 255–265. | Article | PubMed |
4. Waldinger MD, Hengeveld MW, Zwinderman AH, Olivier B. Effect of SSRI antidepressants on ejaculation: a double-blind, randomized, placebo-controlled study with fluoxetine, fluvoxamine, paroxetine, and sertraline. J Clin Psychopharmacol 1998; 18: 274–281. | Article | PubMed | ISI | ChemPort |
5. Kim SC, Seo KK. Efficacy and safety of fluoxetine, sertraline and clomipramine in patients with premature ejaculation: a double-blind, placebo controlled study. J Urol 1998; 159: 425–427. | PubMed | ISI | ChemPort |
6. Hsieh JT et al. In vivo evaluation of serotonergic agents and -adrenergic blockers on premature ejaculation by inhibiting the seminal vesicle pressure response to electrical nerve stimulation. Br J Urol 1998; 82: 237–240. | Article | PubMed |
7. Kim SC, Seo KK, Han JH, Lee MY. Inhibitory effect of serotonergic drugs on contractile response of the rat vas deferens to electrical nerve stimulation: in vivo study. J Urol 2000; 163: 1988–1991. | Article | PubMed |
8. Berendsen HHG, Broekkamp CLE. Comparison of stimulus properties of fluoxetine and 5-HT receptor agonists in a conditioned taste aversion procedure. Eur J Pharmacol 1994; 253: 83–89. | Article | PubMed |
9. Foreman MM, Hall JL, Love RL. The role of the 5-HT2 receptor in the regulation of sexual performance of male rats. Life Sci 1989; 45: 1263–1270. | Article | PubMed | ISI | ChemPort |
10. Ahlenius S et al. Effects of a new type of 5-HT receptor agonist on male rat sexual behavior. Pharmacol Biochem Behav 1981; 15: 785–792. | Article | PubMed | ISI | ChemPort |
11. Waldinger MD et al. Premature ejaculation and serotonergic antidepressants-induced delayed ejaculation: the involvement of the serotonergic system. Behav Br Res 1998; 92: 111–118. | Article |
12. Kihara K, de Groat W. Sympathetic efferent pathways projecting bilaterally to the vas deferens in the rat. Anat Res 1997; 248: 291–299. | Article |
13. Learmonth JR. A contribution to the neurophysiology of the urinary bladder in man. Brain 1931; 54: 147–176.
14. Coffey DS. Androgen action and the sex accessory tissues. In: Knobil E, Neil JD, Ewing LL, Green wald GS, Markert CL, Pfaff DW (ed) . The Physiology of Reproduction. Raven Press: New York, 1988, pp 1081–1112.
15. Waldinger MD et al. The selective serotonin re-take inhibitors fluvoxamine and paroxetine differ in sexual inhibitory effects after chronic treatment. Psychopharmacology 2002; 160: 283–289. | Article | PubMed | ISI | ChemPort |
16. Mos J et al. A comparison of the effects of different serotonin reuptake blockers on sexual behaviour of the male rat. Eur Neuropsychopharmacol 1999; 9: 123–135. | Article | PubMed | ISI | ChemPort |
17. Benson GS. Erection, emission, and ejaculation: physiologic mechanisms. In: Lipshultz LI, Howards SS (ed). 3rd edn. Mosby: St Louis, 1997, pp 155–172.
18. Adenekan O. Evidence of 1-adrenoceptor subtype predominance in the rat seminal vesicle. J Pharm Pharmacol 1989; 41: 277–278. | PubMed |
19. Hsieh JT, Liu SP, Hsieh CS, Cheng JT. An in vivo evaluation of the therapeutic potential of sympatholytic agents on premature ejaculation. Br J Urol 1999; 84: 503–506. | Article |
20. Cavallini G. Alpha-1 blockade pharmacotherapy in primitive psychogenic premature ejaculation resistant to psychotherapy. Eur Urol 1995; 28: 126–130. | PubMed | ChemPort |

Acknowledgements

This work was supported by Korea Research Foundation Grant (KRF-2000-042-F00077). We thank Dr Seong-Joo Jeong for his critical review and assistance in the preparation of this manuscript.