| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Pelvipharm Laboratories,2 CNRS, 91190 Gif-sur-Yvette, France
AP-HP,3 Neuro-Uro-Andrology, Department of Physical Medicine and Rehabilitation, Raymond Poincaré Hospital, 92380 Garches, France
University of Versailles Saint Quentin,4 UFR Paris Ile-de-France Ouest, 78000 Versailles, France
ABSTRACT
Synchronized activation of autonomic and somatic divisions of the nervous system respectively destined to the seminal tract, including the bladder neck and the pelvi-perineal striated musculature, is necessary for anterograde ejaculation. We aimed at investigating the role of intermesenteric nerves (IMNs) in ejaculation in anesthetized rats. Electrical stimulation of intact IMNs and distal and proximal stumps of the sectioned IMN were tested in isoflurane-anesthetized male rats. Electrical stimulation of the intact IMN was also applied to rats with acute spinal transection at the T8 level. The effects of IMN electrical stimulation on emission and expulsion phases of ejaculation were evaluated by measuring seminal vesicle pressure (SVP) and bulbospongiosus (BS) muscle contractions, respectively. IMN electrical stimulation could induce SVP increase and rhythmic contractions of BS muscle concomitantly with expulsion of the seminal plug. When compared with intact IMN electrical stimulation, the occurrence of ejaculation and rhythmic BS muscle contractions, but not SVP increase, was reduced in response to electrical stimulation of the distal stump of the sectioned IMN. In comparison to intact IMN electrical stimulation, the occurrence of ejaculation and rhythmic BS muscle contractions was not significantly modified, whereas the increase in SVP was diminished when the proximal stump of the sectioned IMN was stimulated. Spinalization abolished ejaculation and rhythmic BS muscle contraction but did not impair SVP increase. It is concluded that both afferents conveyed by IMN and relaying supraspinally and efferents of IMN are involved in IMN electrical stimulation-induced ejaculation. We propose that the IMN electrical stimulation paradigm can be used to investigate physiological and pharmacologic aspects of ejaculation.
bulbospongiosus muscle, central nervous system, ejaculation, intermesenteric nerve, male sexual function, rat, seminal vesicles
Ejaculation consists of the coordinated succession of distinct physiological events. Closure of the bladder neck, to prevent flow of semen backward in the bladder from the prostatic urethra, precedes and goes with the emission phase (i.e., secretion by seminal vesicles, prostate, and ampullary vasa deferentia content, which is poured into the prostatic urethra when these organs contract) [1, 2]. Forceful expulsion of seminal material to the urethral meatus is then caused by rhythmic contractions of the pelvic and perineal striated muscles, of which the bulbospongiosus (BS) muscle plays a primary role [3]. The occurrence of ejaculation necessitates the processing of somatosensory and visceral sensory information from the different anatomic structures that constitute the male reproductive organs. Coordinated activity of the brain and spinal nuclei controlling the anatomic entities involved in the ejaculatory process is mandatory for efficient expulsion of sperm.
The importance of the peripheral autonomic nervous system in regulating the ejaculatory response is well documented [4, 5]. In the rat, the majority of sympathetic fibers proceed either via the splanchnic nerves or after relaying in the celiac superior mesenteric ganglia via the intermesenteric nerves (IMNs) to the inferior mesenteric ganglia [6]. Emanating from the inferior mesenteric ganglia are the hypogastric nerves (HNs) that end in the pelvic or inferior hypogastric plexus, the major pelvic ganglion in male rats. Within the pelvic plexus, efferent axons from HNs are intermingled with those running in the pelvic nerves (mainly parasympathetic, but also sympathetic efferents coming from the paravertebral sympathetic chain) [7]. Autonomic neural fibers innervating the anatomic structures involved in ejaculation emanate from the pelvic plexus. The dorsal nerve of the penis, a sensory branch of the pudendal nerve, carries impulses to the upper sacral and lower lumbar segments of the spinal cord from sensory receptors harbored in the penile skin, prepuce, and glans [8, 9]. A second afferent pathway is made up of fibers traveling in the HNs and IMNs and entering the spinal cord via thoracolumbar dorsal roots [10]. In rats as well as in humans, stimulation of sensory afferents, originating in sex organs and conveyed to the lumbosacral spinal cord via the sensory branch of the pudendal nerve, has been shown to provoke the expulsion reflex, involving rhythmic contractions of ischiocavernosus and BS muscles [11–14]. The role of afferents conveyed to the spinal cord via the HNs and IMNs is, however, unclear.
The present study was undertaken to clarify the role of IMNs in ejaculation and to further explore the suitability of using electrical stimulation of peripheral nerves to elicit ejaculation in anesthetized rats.
All animal experiments were carried out in accordance with the European Community Council Directive (86/609/EEC) on the use of laboratory animals. All efforts were made to minimize animal suffering and to reduce the number of animals used.
Sexually naïve adult male rats weighing 200–300 g (Charles River, L'Arbresle, France) were anesthetized with isoflurane (2.5% during surgery; 1.1%–1.4% during the test period). Their body temperature was maintained at 37°C with a homeothermic blanket during the experiment.
The left seminal vesicle was exposed via celiotomy, and a polyethylene catheter (0.5 mm in diameter) filled with heparinized saline (100 IU/ml) was inserted through the apex of the seminal vesicle. The catheter was connected to a T-tube, allowing simultaneous saline perfusion (0.5 µl/min, to prevent the formation of a seminal secretion clot at the tip of the catheter) and seminal vesicle pressure (SVP) determination. Changes in SVP were measured with a pressure transducer (EM 750; Elcomatic, Glasgow, U.K.), amplified (gain, 1000; PCI DAS-1000; DIPSI, Chatillon-sous-Bagneux, France), and digitized (Elphy software; Sadoc, Gif-sur-Yvette, France).
A pair of stainless steel electrodes (32 gauge) was placed within the BS muscle exposed via a perineal incision for recording electromyographic activity (BS EMG). Electrical signals from BS muscles were amplified (DP-301; Warner Instrument Corporation, Phymep, Paris, France; gain, 10 000; low pass, 10 kHz; high pass, 10 Hz) before being digitized.
The IMNs, identified ventrally at the level of the aorta bifurcation or proximal iliac artery, were carefully freed from surrounding connective tissue with the aid of a dissecting microscope. Bipolar platinum stimulating electrodes connected to an electrical stimulator (Biologic SMP 300; Echirolles, France) were placed on IMNs, and the electrode-nerve contact area was filled with mineral oil. Electrical stimulation of the IMNs consisted of 5 square wave pulses (1-msec duration, 6 V, 60 Hz) applied for 30 sec. Each rat received five IMN electrical stimulations, and each stimulation was separated by a 15-min interval.
For T8 spinalization, the skin and muscles over the midthoracic vertebrae were incised, and small retractors were used to separate the muscles overlying the spinous processes of the T6-T8 vertebrae. The T8 spinal cord was exposed through a laminectomy of the T7-T8 vertebrae. The dura was incised, and a complete transversal section of the underlying T8 spinal cord was performed. Completeness of the section was verified by exposing the transverse surface of the proximal and distal stumps of the transected spinal cord.
The occurrence of seminal plug expulsion and rhythmic BS muscle contractions following IMN electrical stimulation was determined in each experimental condition. Interexperimental condition comparisons were performed by the Fisher exact test with the Holm correction for multiple comparisons. Values of P indicated thereafter are corrected values.
The maximal amplitude of SVP increase in response to IMN electrical stimulation (i.e., maximal value reached upon electrical stimulation minus baseline value before electrical stimulation) was averaged for each rat and then for each experimental condition. One-way analysis of variance, followed, whenever P < 0.05, by the Newman-Keuls post hoc test was performed for interexperimental condition comparisons.
Electrical Stimulation of the Intact IMN
Electrical stimulation of the intact IMN induced a rise in SVP, often followed by the rhythmic activity of the BS muscle with or without concomitant seminal plug expulsion (Fig. 1A). It is to be noted that plug expulsion was always accompanied by an SVP increase and intense BS muscle contractions. Thereafter, when referring to seminal plug expulsion, it is implied that the SVP rises and that the rhythmic BS muscle contractions were present. SVP increases occurred immediately at the onset of IMN electrical stimulation and were characterized by a steep rise, followed by a progressive fall starting before the end of electrical stimulation. The rhythmic activity of the BS muscle was characterized by coordinated contractions that were identified by bursts of contraction on EMG recordings that occurred 35 ± 8 sec after the end of IMN electrical stimulation. Contractile responses of the seminal vesicle and BS muscle were accompanied by tumescence of the penile shaft and sometimes (47% of total IMN electrical stimulations) by anteroflexions of the penis due to a straightening of the penile body (flips); whenever ejaculation occurred, intense erection of the glans (cups) was visualized.
|
In a series of experiments involving five repeated electrical stimulations of intact IMNs in 20 rats, plug expulsion occurred 29 times in 16 rats (Table 1). In addition, rhythmic BS muscle contractions without plug expulsion were observed 14 times in 10 rats (Table 1). Three rats displayed neither plug expulsion nor BS muscle contractions in response to the five IMN electrical stimulations applied. A careful examination of SVP recordings did not reveal any significant difference in the pattern and amplitude of SVP increases, whether they were followed by plug expulsion or not. Therefore, values of SVP amplitude were determined and averaged independently of plug expulsion occurrence. The mean value of maximal SVP amplitude measured in rats with intact IMNs was 89 ± 10 mm Hg (Fig. 2).
|
|
We hypothesized that efferent and afferent fibers running in the IMNs played a role in the observed responses to IMN electrical stimulation. To decipher between their respective roles, the IMN was transected, and either the distal stump (to assess the role of efferent neural fibers) or the proximal stump (to assess the role of afferent neural fibers) was stimulated with the same electrical parameters as the intact IMN.
Electrical Stimulation of the Distal Stump of the Sectioned IMN
Electrical stimulation of the distal stump of the sectioned IMN was performed in 10 rats for a total of 45 electrical stimulations (the last two and three electrical stimulations could not be performed in two rats for technical reasons). The occurrence of plug expulsion and rhythmic BS muscle contractions was less frequent in this group of rats when compared with animals having an intact IMN (Fisher exact test, P < 0.01; Table 1). The maximal amplitude of SVP increase elicited by electrical stimulation of the distal stump was similar to that obtained in intact IMN electrical stimulation experiments (90 ± 18 vs. 89 ± 10 mm Hg, respectively, Fig. 2).
Electrical Stimulation of the Proximal Stump of the Sectioned IMN
Electrical stimulation of the proximal stump of the sectioned IMN was performed in five rats with a total of 25 electrical stimulations. An example of a recording (Fig. 1B) shows the absence of SVP phasic increase and the presence of BS muscle rhythmic contractions in response to electrical stimulation of the proximal stump of the sectioned IMN. Frequency of plug expulsion was not significantly decreased when compared with the intact IMN electrical stimulation group (Fisher exact test, P > 0.025; Table 1). Conversely, occurrence of rhythmic activity of BS muscle was numerically higher than in intact IMN electrical stimulation, although the difference did not reach statistical significance (Fisher exact test, P = 0.27; Table 1). In comparison to electrical stimulation of the distal stump of the sectioned IMN, the proportion of rats displaying plug expulsion following electrical stimulation of the proximal stump was not significantly different (Fisher exact test, P = 0.61; Table 1), whereas the proportion of rats exhibiting rhythmic BS muscle contractions was significantly higher (Fisher exact test, P < 0.017; Table 1). The measurement of SVP during electrical stimulation of the proximal stump of the sectioned IMN showed a dramatically decreased maximal amplitude (9 ± 8 mm Hg) when compared with the intact IMN and distal stump-stimulated groups (ANOVA plus the Newman-Keuls post hoc test, P < 0.001; Fig. 2).
To determine whether afferent inputs are integrated at the spinal or supraspinal level to induce BS muscle contractions, we have performed electrical stimulation of the intact IMN in 16 acutely spinalized rats at the T8 level (80 electrical stimulations in total).
Electrical Stimulation of the Intact IMN in T8 Spinalized Rats
Both plug expulsion and rhythmic BS muscle contractions were abolished in T8 spinalized rats (see example in Fig. 1C), although emission of liquid seminal material occurred at the urethral meatus in 10 of 16 rats during the first IMN electrical stimulation. The maximal amplitude of SVP increase induced by intact IMN electrical stimulation in T8 spinalized rats (87 ± 5 mm Hg) was unchanged in comparison to that observed in rats with intact spinal cord (Newman-Keuls post hoc test, P > 0.05; Fig. 2).
The ability of IMN electrical stimulation to induce ejaculation in anesthetized rats has not been previously reported, although the role of the sympathetic pathway, including IMNs and HNs, in the control of bladder neck closure, contraction of seminal vesicles, and seminal fluid secretion has been demonstrated in dogs and rats [15–17]. The IMN connects the celiac superior mesenteric ganglion to the inferior mesenteric ganglion. At the level of the inferior mesenteric ganglion, the IMN projects to the lumbar colonic nerves and HNs. The IMN/HN pathway is a preferential route for efferent sympathetic fiber issued from the thoracolumbar levels of the spinal cord destined to the pelvis, including the seminal tract. It is also a preferential pathway for the corresponding viscerofugal afferent fiber en route to the thoracolumbar levels of the spinal cord [10, 18]. The present results show that, under our experimental conditions, both efferents and afferents traveling throughout the IMN likely participate in electrically induced ejaculation, by controlling both emission and expulsion phases of ejaculation. Upon electrical stimulation of the proximal stump of the sectioned IMN, the emission phase of ejaculation was severely impaired, as indicated by weak contractions of the seminal vesicle. This very likely explains the slight, although not significant, decrease in the proportion of proximal stump IMN electrical stimulation resulting in plug expulsion, despite unchanged frequency of rhythmic BS muscle contractions (i.e., expulsion phase). Conversely, emission took place when the distal stump of the sectioned IMN was stimulated, but the lack of afferent inputs was very likely responsible for the lack of rhythmic BS muscle contractions. The impairment of the expulsion phase inevitably led to a lower incidence of plug expulsion. The highest rate of plug expulsion was obtained upon electrical stimulation of intact IMNs, likely because it caused emission by direct stimulation of efferent fiber to the seminal tract and expulsion by recruiting afferents from the seminal tract.
In rats with acute T8 spinalization, plug expulsion and rhythmic BS muscle contractions induced by IMN electrical stimulations were abolished, whereas contractions of the seminal vesicle were unaffected. It is noteworthy that emission of opalescent liquid at the urethral meatus (dribbling ejaculation) was observed in some of the acutely spinalized rats. The absence of plug expulsion in this group of rats can therefore be explained by impairment of the expulsion phase, underlined by the lack of rhythmic BS muscle contractions. These observations lead us to suggest that under our experimental conditions, the neural circuitry involved in the control of the expulsion phase of ejaculation elicited by IMN electrical stimulation includes an ascending pathway reaching cerebral areas, which activate, via descending projections, the spinal motoneurons commanding the BS muscle located in the dorsomedial nucleus of the lumbosacral spinal cord [19]. Whether activation of lumbosacral motoneurons is produced by descending cerebral projections directly connected to these neurons or connected to the spinal ejaculatory center (lumbar spinothalamic cells [LSt]) that, in turn, projects on lumbosacral motoneurons [20] remains to be elucidated.
A segmental reflex circuit for striated perineal muscles (including BS muscle) exists in the lumbosacral spinal cord (L6-S1 in rats). The primary sensory input to this segmental reflex circuit, and probably to the LSt cells, consists of the dorsal nerve of the penis and, proximally, of the sensory branch of the pudendal nerve, which drives sensory information originating in the perigenital area, in the erectile tissue, and in the urethral mucosa [8, 21]. Activation of pudendal nerve afferents induces reflex discharge of lumbosacral motoneurons as well as rhythmic contractions of BS muscle [11, 21, 22]. This polysynaptic reflex, which includes lumbosacral interneurons, is entirely handled in the spinal cord, although it is modulated by inhibitory descending projections originating in the gigantocellular nucleus of the medulla oblongata [11]. In contrast, another pathway, partly identified in the present study, comprises viscerofugal afferents projecting from the seminal tract in the HNs and further into the IMNs, which enter the thoracolumbar spinal cord and reach the supraspinal site(s). This pathway plays an activating role on the expulsion phase of ejaculation, possibly by acting on LSt cells connected to lumbosacral motoneurons. Taken together, these data indicate the existence of two different neural networks involved in the expulsion phase of ejaculation, which comprise two distinct afferent arms and very likely distinct supraspinal sites with opposite modality of action on the control of the expulsion response. Both networks share, as common spinal elements, lumbosacral motoneurons innervating BS muscle and possibly LSt cells. One may suggest that the distinct elements of these two networks, which can work independently from each other, are part of a redundant system that ensures execution of the expulsion phase of ejaculation, essential for the occurrence of effective anterograde ejaculation and thereby procreation.
In men with complete spinal cord lesion above T9, electrical stimulation of the hypogastric plexus, where the HN originates, led to dribbling ejaculation with no report of pelvic contractions [23]. Interestingly, the authors reported that in one patient, with incomplete spinal cord (C7) lesion and tactile and proprioceptive sensitivity maintained over his whole body, contraction of lower abdominal muscles and flexion of the hips occurred as a reflex to the stimulation. This indicates that a partial connection between the brain and spinal cord is necessary for the reflex activation of pelvic muscles in response to stimulation of the hypogastric plexus. Alternatively, ejaculation can also be induced in men with complete spinal cord lesion (above lower thoracic segments) by applying vibratory stimulation to the penis. With this procedure, reflex contractions of the BS and ischiocavernosus muscles are observed concomitantly with the expulsion of sperm and are inhibited by local anesthesia of the dorsal nerve of the penis [24]. From the above data collected in men with spinal cord injury, one may suggest that two distinct afferent pathways can trigger the expulsion phase of ejaculation: one includes viscerofugal afferents traveling throughout the sympathetic pathway and necessitates a supraspinal relay, and the other one includes penile afferents and is entirely handled in the spinal cord. This comes into line with our findings in rats.
The use of isoflurane as the anesthetic was decisive in this study, as rhythmic contractions of BS muscles and plug expulsions could not be induced by IMN electrical stimulation in rats anesthetized with urethane (1.2 g/kg i.p.), pentobarbital (60 mg/kg i.p.), or ketamine/xylazine (90 and 9 mg/kg, respectively, i.m.) (data not shown). A clear advantage of inhaled anesthetics such as isoflurane is the possibility to modulate the level of anesthesia on demand throughout the experiment. Isoflurane anesthesia could be maintained high during surgery (up to 3.5%) and lowered when performing electrical stimulation (down to 1.1%–1.3%). Increasing isoflurane anesthesia beyond 2% during IMN electrical stimulation abolished the occurrence of expulsion reflex and plug expulsion (data not shown). Another potential advantage of isoflurane might rely on its specific mechanism of action. It is worth noting that isoflurane is known to interact with the serotonergic system [25], which plays a key role in the control of ejaculation [26]. Nevertheless, the reason why such an interaction could promote the occurrence of ejaculation under our experimental conditions needs further investigation.
In conclusion, we have demonstrated that IMN electrical stimulation can elicit ejaculation in anesthetized rats and that viscerofugal afferents running in IMNs and relaying supraspinally play a role in the expulsion phase of ejaculation, whereas sympathetic efferents of the IMN command the emission phase of ejaculation (Fig. 3). This experimental paradigm may help improve our understanding of the central and peripheral physiology and pharmacology of ejaculation.
|
ACKNOWLEDGMENTS
The authors wish to thank S. Caisey, S. Compagnie, and M. Laurin for their excellent technical assistance as well as A. Wohlhuter for proofreading.
Correspondence: 1François Giuliano, Neuro-Uro-Andrology, Department of Physical Medicine and Rehabilitation, Raymond Poincaré Hospital, 92380 Garches, France. FAX: 33 1 4710 4443; e-mail: giuliano{at}cyber-sante.org
Received: 16 February 2007.
First decision: 5 April 2007.
Accepted: 18 June 2007.
REFERENCES
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
