Rhythmic Motor Patterns Accompanying Ejaculation in Spinal Cord-Transected Male Rats

ORIGINAL ARTICLE Rhythmic motor patterns accompanying ejaculation in spinal cord-transected male rats

M Carro-Jua´rez1, G Rodrı´guez-Manzo2, M de Lourdes Rodrı´guez Pen˜a1 and MA´ Franco1

A spinal pattern generator controls the ejaculatory response. Activation of this spinal generator elicits rhythmic motor patterns of the striated musculature that surrounds the genital tract that contributes to the expulsion of seminal secretions. In the present study, we elicited ejaculation in spinal cord-transected male rats by mechanically stimulating the urethra and registered rhythmic motor patterns in the cremasteric, iliopsoas and pubococcygeus muscles. The rhythmic motor activity recorded in these muscles was compared with that elicited in the bulbospongiosus muscles; the results revealed similarities in the motor parameters among all the muscles. Data of this study, showing the occurrence of rhythmic motor behaviour in the cremasteric, iliopsoas and pubococcygeus muscles during ejaculation, suggest that these muscles might be under the control of the spinal generator for ejaculation.

International Journal of Impotence Research (2014) 26, 191–195; doi:10.1038/ijir.2014.4; published online 20 February 2014 Keywords: ejaculation; pelvic and abdominal striated muscles; rat; rhythmic motor pattern; spinal cord; spinal generator for ejaculation

INTRODUCTION SGE’s initial activation by genital inputs, multiple rhythm- Ejaculation is the physiological process that describes the generating cores of the ejaculation circuit could be activated expulsion of semen from the urethra and consists of two different simultaneously within the lumbosacral spinal cord, initiating 4 phases, an emissive phase and an ejective phase.1–4 The emissive multiple motor patterns. Electromyographic (EMG) studies of 11 12 phase implies the closure of the bladder neck and the contraction the pudendo-pudendal, the bulbocavernosus and the glans- 13 of the sexual accessory glands, including the seminal vesicles, vasal, urethromuscular, cavernoso-urethral and abdominal- 14 prostate, vas deferens and coagulant glands.1–4 The ejective phase genital reﬂexes reported to participate in ejaculation, partially consists of the forceful ejection of semen from the urethral support these hypotheses and show that, after mechanical meatus, caused by the rhythmic contraction of the striated genital stimulation of genital structures, several rhythmic patterns of muscles.1–4 The ejective phase of ejaculation is considered the muscular activity occur. However, the whole striated muscular main indicator of ejaculation.4 activity observed during ejaculation, which could be considered as Experimental studies have provided the physiological evidence the entire somatic component of ejaculation, has not been fully for the involvement of a spinal pattern generator in the control of characterised. ejaculation.5–8 Activation of this spinal generator for ejaculation A general picture about the participation of striated muscles (SGE), by stimulation of genital structures in anaesthetised and and their neural control in ejaculation is now available. Thus, it has spinal cord-transected animals, elicits the complete sequence of been demonstrated that genital striated muscles such as the genital activities associated with seminal ejection, which includes levator ani, bulbospongiosus, ischiocavernosus and urethralis as the potent and rhythmic expulsion of seminal secretions, well as of the sphincters all participate in the ejective phase of movements and erections of the penis and the rhythmic ejaculation.4,5,13,15 However, other pelvic and abdominal muscles contraction of the genital striated muscles.4,9,10 Although it is are suggested to intervene in the ejaculatory phenomenon. This well known that the SGE mediates the genital autonomic and would be the case of the cremasteric (Cm) and the somatic outﬂow during the two phases of ejaculation and pubococcygeus (Pbc) muscles,16,17 as well as of muscles of the integrates sensory inputs necessary to trigger this sexual posterior abdominal wall, such as the iliopsoas (Ilps) muscles. It response, evidence regarding the neural mechanisms and has been proposed that these muscles contract rhythmically to substrates of this spinal network is scarce.4 lead to intense pelvic thrusting, resulting in deep deposition of Our recent hypotheses on the spinal control of ejaculation seminal secretions.18 suggest that the SGE has a modular structure and to attain In the present work, we hypothesised that these abdominal ejaculation, one major ejaculation generating core of this pattern muscles would exhibit rhythmic activity during the ejaculatory generator, located in the upper segments of the lumbar cord, response, simultaneous to the one exhibited by the genital drives the SGE pacemaker activity towards multiple rhythm- muscles already described, supporting the notion that they generating cores of the ejaculation circuit.4 As a consequence of participate in the ejaculatory response. To test this hypothesis,

1Laboratorio de Comportamiento Reproductivo, Escuela de Medicina Veterinaria y Zootecnia, Universidad Autońoma de Tlaxcala, Tlaxcala, Me´xico and 2Departamento de Farmacobiologıá, Cinvestav-SedeSur, Calzada de los Tenorios 235, Col Granjas Coapa, Delegacio´ n Tlalpan, Me´xico D.F., Mexico City, Me´xico. Correspondence: Dr M Carro-Jua´rez, Laboratorio de Comportamiento Reproductivo, Escuela de Medicina Veterinaria y Zootecnia, Universidad Autońoma de Tlaxcala, AP 484, Huamantla Tlax domicilio conocido, Tlaxcala 9000, Mexico. E-mail: [email protected] Received 6 August 2013; revised 10 December 2013; accepted 9 January 2014; published online 20 February 2014 Rhythmic motor activity of ejaculation M Carro-Jua´rez et al 192 we used the fictive ejaculation model in spinal cord-transected muscles. This muscle was selected as a monitor of ejaculatory activity, male rats and evoked the reflexive activation of the selected owing to its superficial position in the perineum and given that it muscles by the mechanical stimulation of the urethra (5, for discharges rhythmically during ejaculation, in synchrony with all genital review; 19). The fictive ejaculation model permits the recording muscles. Thereafter, the RMPE were activated by mechanically stimulating and visualisation of the rhythmic motor pattern of ejaculation the pelvic urethra with saline solution. Saline was administered with a syringe pump, through a PE-50 catheter (0.965 mm OD) inserted into the (RMPE) in separated striated muscles that is accompanied by pelvic urethra via a bladder incision, at a rate of 200 ml min À 1 for 10 s, while complex pelvic activity including phasic and strong penile erec- occluding the penis meatus to achieve an intraurethral pressure, which tions, as well as penile movements, followed by the potent expul- ranged from 20–30 mm Hg. The RMPE was activated in the Bsp muscle by sion of urethral contents. urethral stimulation at 3-min intervals and the EMG activity of the other selected muscles, if present, was simultaneously monitored searching for its rhythmic activation during the ejaculatory response. At this moment, MATERIALS AND METHODS the stimulation protocol was finished. Animals Sexually experienced male Wistar rats averaging 300–350 g body weight Drugs were used. Animals were housed by groups (four rats per cage) under an Urethane hydrochloride was purchased from Sigma Chemical (St Louis, inverted light/darkness cycle 12:12 h, at 22 1C, and with free access to food MO, USA) and dissolved in distilled water. and water. The Local Committee of Ethics on Animal Experimentation approved all experimental procedures, which followed the regulations established in the Mexican official norm for the use and care of laboratory Data analysis animals ‘NOM-062-ZOO-1999’. EMG activity was recorded differentially, amplified and filtered (1000 Â , 0.1–1 kHz bandpass) in a Poliview Data Acquisition System (Grass). The parameters recorded in the muscular trains during ejaculatory responses Groups were: latency to the appearance of the motor response and the number Animals were divided into four groups (N ¼ 4, each). Group 1 was utilised and frequency of EMG bursts. All numerical values were expressed as to record the EMG activity of the Bsp muscle, elicited by the mechanical means±s.e.m. Mean values were calculated for each individual animal and stimulation of the urethra expressed as the RMPE, and served as the paired comparisons between the control group and each of the control group. Animals in groups 2–4 were employed to register the experimental groups were conducted by means of the Mann–Whitney probable expression of the RMPE in the Cm (G2), Ilps (G3) and Pbc (G4) U-test. A Po0.05 was considered to be statistically significant. The Stat muscles in response to the mechanical stimulation of the urethra. program (SigmaStat 3.5, Jandell Scientific Corp., San Raphael, CA, USA) was used for all statistical analyses. Sexual behaviour observations To provide sexual experience, male rats received five sexual behaviour RESULTS tests with receptive females. Female receptivity was induced by the sequential subcutaneous administration of oestradiol valerianate (4 mg per The ejaculatory motor pattern of spinal cord-transected male rats rat, s.c.) followed 44 h later by progesterone (2 mg per animal, s.c.). evoked by urethral stimulation and registered in the Bsp muscle, Behavioural observations were conducted 4 h after progesterone admin- consisted of a first rhythmic motor train followed by an after- istration and 2 h after the onset of darkness. Males were introduced into a discharge train (see Figure 1). Bsp ejaculatory rhythmic motor cylindrical observation cage and a 5 min adaptation period was allowed. patterns contributed to the potent expulsion of the urethral Then, a receptive stimulus female was introduced into the cage and contents, promoted by the expression of phasic penile erections copulation was permitted for a 25-min period. Sexually active male rats and penile movements, and exerted pressure on the base of the exhibiting ejaculation latencies of less than 15 min in the last three sessions were considered as sexually experienced male rats and selected penis, promoting penile rigidity. These ejaculation-associated for the study.

General surgical procedures All animals were anaesthetised with urethane (0.7 g kg À 1, intraperito- neally). The adequacy of anaesthesia was assessed by the absence of a withdrawal reflex after noxious paw pinch. Once anaesthetised, animals were placed on a surgical table in a dorsal supine position and after a small longitudinal incision under the skin of the testicular region the Cm muscle was identified, located immediately posterior to the muscular transition region, between the oblique internal and transverse abdominal muscles where the Cm muscle arises.20 The Bsp muscles were found after a surgical incision on the perineum, close to the penis, as previously described.4,5 The Pbc muscle was identified within the pelvic floor, close to the tail where it arises, by following its fleshy wide portion.17 Finally, into the abdominal cavity and after a medial longitudinal laparatomy, the Ilps muscle was dorsally located after the displacement of abdominal viscera, following the iliac vein close to the caudal portion of the kidney, just in the medial fleshy portion of the muscle.20–22 Two platinum wires (Grass Astro-Med, Grass instrument division, Astro-medic Inc., West Warwick, RI, USA, 2003) were inserted into one of the selected muscles (Cm, Pbc or Ilps) and another pair into the Bsp muscle to simultaneously record the EMG activity, which was registered on a polygraph (Grass M7). At the end of the surgery the spinal cord was blunt transected at the T6 spinal cord level. Figure 1. Polygraphic tracing showing typical rhythmic motor patterns during ejaculation elicited by mechanical stimulation of Activation and recording of the selected muscles the urethra in the spinal cord-transected male rat. The rhythmic Immediately after spinal cord transection, the RMPE is spontaneously motor trains of the bulbospongiosus muscle are constituted by two expressed in the genital muscles with a mean latency of 1–3 min.5,7 In main components including a first ejaculatory motor train followed order to assess the ability of the spinal cord to produce the activation of by an after-discharge component (a), whereas the rhythmic motor the selected muscles and for comparison purposes, after spinalisation, one trains registered in the cremaster (b), Ilps (c) and Pbc (d) muscles spontaneous RMPE was allowed to express and it was recorded in the Bsp lacked the after-discharge component. Calibration bar 10 s, 50 mV.

International Journal of Impotence Research (2014), 191 – 195 & 2014 Macmillan Publishers Limited Rhythmic motor activity of ejaculation M Carro-Jua´rez et al 193 penile responses were observed simultaneously to the recording exhibited a mean response latency of 10±6.68 s, a mean number of the rhythmic contractions of Bsp muscles, during ejection of of 8.25±0.85 discharges and a frequency of discharge of urethral contents. 1.32±0.13 Hz. The Ilps muscle presented motor trains with a The Cm, Pbc and Ilps muscles were all activated by the latency of response of 24.3±11.09 s, a mean number of mechanical stimulation of the urethra and exhibited rhythmic 7.25±1.49 discharges and a frequency of discharge of motor trains during the ejaculatory response. The activation of 1.1±0.04 Hz (Figure 2). each of these muscles was simultaneous to that of the Bsp muscle. The EMG responses of the Cm muscle showed some After-discharge activity was only observed in the Bsp muscle; the particularities when compared with the rest of the analysed Cm, Pbc and Ilps muscles lacked this motor component. The muscles. The Cm muscle displayed constant periods of basal EMG rhythmic motor trains exhibited by the Cm, Pbc and Ilps muscles tonic activity, and the rhythmic motor trains evoked by urethral were similar to those registered in the Bsp muscles. The analysis of stimulation overlapped with this tonic activity. Once the urethral the parameters of these motor trains, that is, latency to response, stimulation-evoked rhythmic activity of the Cm finished, the basal number and frequency of motor discharges of the ejaculatory tonic activity remained for several minutes (Figure 1). The trains registered in the Cm, Pbc, Ilps and Bsp muscles showed no rhythmic activity of the Cm at ejaculation did not produce statistically significant differences among muscles (see Figure 2). muscular movements with enough force to directly influence the The Bsp muscles displayed rhythmic motor trains during ejacula- expulsion of the urethral contents or the displacement of the tion with a mean latency of response of 10.5±2.09 s and exhibited testes towards the pelvic or the abdominal cavities. motor trains that averaged 7.5±0.64 ejaculatory discharges, with The expression of the rhythmic motor pattern in the Pbc a frequency of discharge of 1.12±0.09 Hz. The Cm muscle muscles during ejaculation did not directly influence the penile contracted rhythmically with a mean latency to response of events, observed during expulsion of urethral contents, but its 13.25±3.35 s, a mean number of 10.25±1.54 discharges and with reflexive rhythmic contraction exerted a sustained movement a mean frequency of discharge of 0.98±0.01 Hz. The Pbc muscle against the base of the urinary bladder. Finally, at ejaculation no noticeable movements of the IIps muscles upon the genital structures were detected. 40 15 35 30 DISCUSSION 25 10 Findings of the present study in spinal cord-transected male rats 20 show that at ejaculation, rhythmic motor trains are switched on in 15 5 the Cm, Ilps and Pbc muscles that are similar to those registered in

Latency (sec) 10 5 the striated muscles associated with the genital tract, the Bsp 0 Number of discharges 0 muscle in this case. The rhythmic motor trains of the striated

Bsp Crm Pbc IlPs Bsp Crm Pbc IlPs pelvic and abdominal muscles here described were simulta- 35 neously evoked by mechanical stimulation of the urethra, without 30 statistically signiﬁcant variations in the parameters of their motor 25 trains. 20 Mechanical stimulation of the urethra is able to activate the SGE 15 to elicit rhythmic motor patterns in the bulboespongiosus,

frequency 10 isquiocavernosus, uretralis and levator ani muscles, which have 4,19,23 of discharge (Hz) 5 been identified to participate in the ejaculatory response. 0 Urethral stimulation recruits urethral primary inputs carried in Bsp Crm Pbc IlPs either the pudendal or pelvic nerves to different segments of the Figure 2. Specific parameters of the rhythmic motor trains lumbar spinal cord, contributing with excitatory projections to the 19 registered in the bulbospongiosus, Cm, Ilps and Pbc muscles in SGE pathways. Data of the present study show that mechanical the spinal cord-transected male rat. Mann–Whitney U-test, non stimulation of the urethra evokes rhythmic motor trains not only significant. in the muscles associated with the genital tract, such as the Bsp

Figure 3. Schematic representation of the spinal intersegmental reﬂex circuit for ejaculation. During ejaculation, sensory information from genital inputs (Gi) converge onto the interneurones conforming the SGE provoking its activation, which in turn switches on rhythmic motor patterns after targeting cremasteric (CmM), iliopsoas (IlpsM), pubococcygeus (PbcM) and bulbospongiosus (BspM) motoneurons, facilitating some postural arrangements necessary for successful ejaculation. T6, thoracic 6; ScT, spinal cord transection; L1–L3, lumbar 1–lumbar 3; L3–L4, lumbar 3–lumbar 4; L5–S1, lumbar 5–sacral 1.

& 2014 Macmillan Publishers Limited International Journal of Impotence Research (2014), 191 – 195 Rhythmic motor activity of ejaculation M Carro-Jua´rez et al 194 muscle, but also in muscles of the abdominal walls such as the segments,31 and the Bsp and Pbc muscles are innervated by Cm, Pbc and Ilps. The fact that the mechanical stimulation of the motoneurons located in the ventral nucleus of lamina IX, and in urethra acts as a common stimulus to evoke rhythmic behaviour the dorsolateral and dorsomedial spinal nucleus at the L5–S1 in all muscles here studied, suggests that sensory information spinal cord segments, respectively.17,32 On the basis of the from the urethra converges into the spinal neuronal networks for location and the neuroanatomical connections of the spinal ejaculation, to generate the rhythmic behaviour of several groups centres for ejaculation, within the medial portion of the lumbar of striated muscles. enlargement of the male rat, we propose that at ejaculation the Ejaculation is a physiological response that involves highly SGE targets simultaneously upper and lower lumbar and sacral synchronised striated motor patterns that participate in seminal segments of the cord, contributing to the control of synergistic ejection to ensure fertilisation.4 Pioneer studies on the spinal rhythmic actions of the Cm, Ilps, Pbc and Bsp muscles (Figure 3). control of ejaculatory mechanisms reported that rhythmic Thus, the ejective phase of ejaculation could be considered to be movements occur in the anogenital region of males, provoking under a multisegmental spinal cord control. In line with this that the penis becomes longer, thicker and protruded, completing notion, the existence of an intersegmental reflex circuit for penile erection and that this genital activity is accompanied by the ejaculation has been demonstrated in normal and spinal cord- discharge of semen.5,7,23,24 This pattern of rhythmic movements in lesioned men, which allows the simultaneous activation of genital spinal cord-transected animals bears significant and striking muscles. This intersegmental circuit permits lumbar and sacral resemblances to those seen in spinally intact animals; however, sensory inputs from its corresponding dermatomes to reach, via it was reported in spinal cord-transected animals that these intraspinal pathways, the Cm and Bsp muscles whose motor nuclei ejaculatory reactions are only registered in the genital muscles are located at different and distant levels of the lumbar and sacral with no evidence of rhythmic motor patterns in additional spinal cord segments.33 However, the complete intraspinal muscles distant to the genital area. Data of the present study circuitry of this intersegmental circuit for ejaculation and all its show that in addition to the activation of the bulbospongiosus targets have not been completely established.33 Findings of the muscles that mediates ejaculation, the mechanical stimulation of present study suggest that during ejaculation, the intersegmental the urethra also activates rhythmic motor patterns in the Cm, Ilps reflex circuit for ejaculation recruits the Cm, Ilps, Pbc and Bsp and Pbc muscles. We have not an explanation for this interesting muscles, among others, to contribute with rhythmic motor finding, but we propose that in spinal cord-transected rats Cm, patterns accompanying the postural arrangements that permit Ilps and Pbc rhythmic contractions elicited by ejaculation might intromission and successful ejaculation such as pelvic thrusting, have to do with postural arrangements employed during movements of the testes and ejection of seminal secretions ejaculation. Thus, for instance, while rhythmic contractions of (Figure 3). Accordingly, the intersegmental reflex circuit could be the bulbospongiosus muscles provoke the intense penile rigidity considered as an integral part of the SGE circuitry, which might and intense penile erections seen during seminal ejection, the mediate the entrance of sensory inputs and the activation of rhythmic contractions of the Pbc could promote pressure changes motor outputs, contributing to the somatic component of in the pelvic floor, and contractions of the Cm muscle might ejaculation. produce elevations of the gubernaculum testis to expel completely all seminal secretions. Finally, Ilps muscles could be in charge of the momentary and deep pelvic thrusting and the CONCLUSION hind leg postural rigidity, that feature long intromissions typical of In conclusion, findings of the present study show that during seminal deposition. Further studies on electrical stimulation of the ejaculation, the mechanical stimulation of the urethra simulta- muscles involved in ejaculation are necessary to support these neously evokes rhythmic motor patterns in the Bsp as well as in suggestions. the Cm, Ilps and Pbc muscles with similar motor parameters. The One of the hypotheses to explain the neural mechanisms rhythmic activity of these muscles could be under the control of underlying rhythmic motor acts such as ejaculation and locomo- the SGE and might participate in the display of the postural tion holds that single pacemaker neurones or a neurones’ arrangements necessary for successful ejaculation. network, named central pattern generator, is able to produce a repetitive, rhythmic output to the muscles involved in the rhythmic behaviour.25–27 Evidence provided in the present study CONFLICT OF INTEREST in spinal cord-transected male rats showing the occurrence of The authors declare no conflict of interest. ejaculatory rhythmic motor behaviour in the Cm, Ilps, Pbc muscles, suggests that these muscles can also be under the control of central pattern generator networks located at the spinal cord level. REFERENCES It could be thought that the SGE that drives the motor outputs to 1 Newman H, Reiss H, Northurp JD. 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& 2014 Macmillan Publishers Limited International Journal of Impotence Research (2014), 191 – 195