T H E ANATOMICAL RECORD 229:203-208 (1991)
The lschiourethralis Muscle of the Rat: Anatomy, Innervation, and Function WILLIAM G. DAIL AND BENJAMIN D. SACHS Department of Anatomy, University of New Mexico School of Medicine, Albuquerque, New Mexico (W.G.D.); and Department of Psychology, University of Connecticut (B.D.S.), Storrs, Connecticut
ABSTRACT The ischiourethralis (IU), a striated perineal muscle presumed to be involved in sexual reflexes, was studied in the rat. The paired muscle arises from the penile crus and the penile bulb and unites in a raphe over the deep dorsal vein of the penis. Retrograde tracing studies show that the muscle is innervated by neurons in the dorsolateral nucleus of the lumbar spinal cord, a pudendal nerve motor nucleus which also innervates the ischiocavernosus muscle. Excision of the IU muscle did not interfere with the ability of males to display normal copulatory behavior, nor did it affect significantly the number and intensity of reflexive erections. It nevertheless remains possible that the IU may contribute to intense glans erection by compressing the deep dorsal vein. The vascular events of penile erection are accompanied by patterned activity of striated penile muscles, including the ischiocavernosi (IC) and the bulbospongiosus (BS). The precise role of the extrinsic muscles may vary according to species and to the stage of copulatory behavior. For example, in addition to its role in the expulsion of semen, the BS muscle of dogs may force blood from the penile bulb to the glans during erection (Hart, 1972). A comparable action has been confirmed for the rat: the BS muscle augments vascular filling of the corpus spongiosum by compressing the penile bulb, and excision of this muscle eliminates full erection of the glans penis and reduces the fertility of mating (Sachs, 1982, 1983; Hart and Melese-d’Hospital, 1983). In the rat, the ischiocavernosus muscles are responsible for penile “flips,” which involve straightening the normal ventrof lexion of the penile body to permit intromission (Sachs, 1982, 1983; Hart and Melese-d’Hospita1, 1983). Similarly, the increased pressure of the corpora cavernosa penis of the dog during intromission is correlated with a n increase in the activity of the ischiocavernosus muscles (Purohit and Beckett, 1976). It has also been proposed that the paired ischiocavernosus muscles may aid in detumescence in the dog (Hart, 1972). Less is known of the role of a band of striated muscle which overlies the deep dorsal vein of the penis in some animals. Houston (1830) first described the muscle in the dog and other mammals and, because of its anatomical relationships, named it the venae compressor dorsalis penis muscle (also see Sisson, 1975). Christensen ( 1954), who renamed the muscle the ischiourethralis (IU), proposed that in the dog, the muscle lessened the free flow of blood through the deep dorsal vein after intromission. Later studies indicated that the IU in the dog contributes to rapid erection and is responsible for the tumescence of the glans penis (Hart and Kitchell, 1966; Hart, 1972). The rat is a frequently used species in which to investigate reproductive function and, while many studc
1991 WILEY-LISS, INC
ies have focused on the BS and the ischiocavernosus muscles (Schr@der,1980; Sachs, 1982, 1983; Hart and Melese-d’Hospita1, 1983; Wallach and Hart, 1983; McKenna and Nadelhaft, 19861, to our knowledge there have been no descriptive or functional studies of the IU in this important animal model. Little is known about the role of venous occlusion in penile erection in the rat, but the position of the IU overlying the deep dorsal penile vein suggests that this muscle may promote penile erection by reducing venous drainage. This hypothetical function is reinforced by the report (Sachs et al., 1989) that section of the motor branch of the pudendal nerve drastically impairs copulatory behavior and prevents rats from displaying even the vascular components of reflexive erection. Although the innervation of IU muscle in the rat remains to be determined, the pudendal nerve is a likely candidate, a s it innervates several other perineal striated muscles associated with sexual or eliminative functions (McKenna and Nadelhaft, 1986).The present investigation provides a n anatomical description of the muscle, identifies the spinal cord location of neurons which innervate it, and determines whether excision of the muscle impairs the mating behavior and erectile potential of rats. MATERIALS AND METHODS Anatomical Studies
The anatomical experiments were conducted on mature male (200 g) Sprague-Dawley rats. Dissection and histological studies were performed on the penes of seven rats which were fixed by perfusion with buffered 10% formalin preceded by flushing with phosphate-
Received May 21, 1990; accepted J u l y 24, 1990. Address reprint requests to William G. D a d , Department of Anatomy, University of New Mexico School of Medicine, Albuquerque, NM 87131.
buffered saline. Paraffin sections were taken through the IU at 6 pm and stained with Gomori’s trichrome to confirm the observations derived from the dissections. An additional three rats were used to determine the location and number of motor neurons which project to the IU. Rats were anesthetized with sodium pentobarbital (30 mgikg i.p.) and prepared for sterile surgery. The IU was exposed by a single incision a t the base of the penis, proximal to the suspensory ligament of the penis. To expose more of the IU, the adjacent ischiocavernosus muscle was partially removed. With the aid of a Picospritzer (General Valve Corp., Fairfield, N J ) , less than 1 pl of a suspension of 2.5% Fluorogold (FG; Flurochrome Inc., Englewood, CO) was injected into each side of the IU. Alternatively, a Hamilton syringe was used to deliver FG to the muscle. The needle tip, which entered the IU near its raphe, was directed laterally beneath the muscle epimysium. Injection of FG resulted in a yellow coloration of the muscle with no overt leakage of the dye. The entry point of the needle was sealed by a cautery. Seven days following the injection of the retrograde tracer, rats were anesthetized and perfused transcardially with phosphate-buffered saline containing procaine hydrochloride, followed by paraformaldehyde (4%) in phosphate buffer. Spinal cord sections L,-L,, identified by the criteria of Rubin and Purves (1980), were removed, rinsed of fixative, and stored overnight in 20% sucrose in phosphate buffer. The L,L, spinal cord segments of the rat contain the alpha motoneurons of the pudendal nerve including those supplying the ischiocavernosus and BS muscles (McKenna and Nadelhaft, 1986). Spinal cord segments were frozen, sectioned at 30 pm, and examined with a fluorescence microscope for labelled cells. A block of tissue containing the IU was also taken to verify the site of the injection of FG. Behavior Studies
Subjects Eight male rats (Long-Evans hooded, 120-150 days of age; 450-600 g) were selected on the basis of having copulated and displayed reflexive penile erections reliably in tests similar to those described below. The rats were randomly assigned to IU excision (IUx) or sham-operated (SHAM) groups ( n = 4 per group). Surgery
After anesthesia induced by i.m. injection of ketamine (50 mgikg) and xylazine ( 4 mgikg), the perigenital area was shaved, and a transverse, 1 cm incision was made in the skin just anterior to the palpable penile flexure. The penile body was then deflected ventrocaudally, and the suspensory ligament was removed to gain access to the IU. For IUx males, the paired muscle was carefully teased away from underlying tissue and cut near the lateral attachments as laterally as possible without disturbing adjacent tissue. Care was also taken to avoid damage to the dorsal penile nerves. For SHAM males, the IU was located but not otherwise disturbed. The incision in t h e skin was then closed with silk sutures, supplemented with a single 7 mm wound clip which was removed under ether anesthesia 6-7 days later. Surgery and recovery were uneventful in all animals.
Behavioral testing
Testing began 9-10 days after surgery. Details of the testing procedures, behavioral variables, and the computer software used for behavioral recording have been fully described elsewhere (Bitran e t al., 1988; Holmes e t al., 1988; Leipheimer and Sachs, 1988). Briefly, for the penile reflex test, a male was placed in supine position with its anterior torso restrained in a loosely fitting cylinder. The penile sheath was retracted to the base of the glans, and the glans and distal penile body were kept exposed by fastening a strip of paper tape, through which a hole had been punched, over the penis. No further stimulation was applied. Events recorded included penile body lengthening, straightening of the flexure (“flips”),and glans erection of three intensities classified as E l , E2, and E 3 or “cup.” Latencies were measured from sheath retraction, and tests continued for 15 minutes after the first reflex, or for 30 minutes if no responses occurred. Copulation was tested 1 week after the reflex test, during the dark phase of the 12:12 Light-Dark cycle in 40 x 21 x 25 cm glass chambers littered with wood shavings. Intromissions (I), mounts without intromission (M), and ejaculations were recorded and counted, and the latency to each behavior was measured from the time of placement of the hormone-induced estrous female into the cage with the male. A key measure of the erectile capacity of males is the intromission ratio (Sachs, 19781, defined as No. Ii(No. M + No. I). (Low intromission ratios can result from a large number of factors, including penile insensitivity, inappropriate orientation of mount, or lack of receptivity of the female. High intromission ratios require all of these and erection of the penile body so that the glans is directed rostrally instead of ventrally [Sachs, 1978, 1982, 19831.) Mating tests continued until the first intromission after the second ejaculation, or, if no ejaculation occurred. for 30 minutes after the first intromission. RESULTS The anatomy of the striated muscles of the r a t penis is depicted in Figure 1.The IC and the BS muscles have been described in detail by McKenna and Nadelhaft (1986) and will not be treated here. The IU is a paired muscle which forms an arch over the deep dorsal vein a t the base of the penile shaft (Fig. 2). The muscle is found a t the region where the pyramidalis muscle covers the pubic symphysis in the rat (Fig. 2). Dissection of the muscle laterally indicates t h a t i t has tendinous origins from the tunica albuginea of the penile crus and penile bulb. An origin from pubic bones was not apparent. Examination of histological sections revealed t h a t the IU is a striated muscle which overlies the dorsal penile arteries and dorsal penile nerves as well as the deep dorsal vein (Fig. 3). Although the IU is closely apposed to the ischiocavernosus, a connective tissue layer intervenes. A midline raphe was apparent in the gross specimens, but was less obvious in the histologic sections. Distally along the penile shaft, smooth muscle is a prominent component of the wall of the deep dorsal vein; however, the vein is relatively thin at the point where it underlies the IU (Fig. 3). At the proximal
205
ISCHIOUKETHRA1,IS MUSCLE OF THE RAT
pyramidalis muscle
...........pyramidalis muscle
...........muscle ischiocavernosus ..bulbospongiosus muscle
ischiourethralis muscle ischiocavernosus muscle deep dorsal vein
..rectum
-
‘ 1 = 3; -&-*.&>Y.g
1
..................shaft of penis
2
Fig. 1, The striated perineal muscles of the rat penis include the IC and the multilobed BS. The dorsal part of the BS passes behind the rectum. The region between the pyramidalis muscle and the base of the penis is shown in detail in Figure 2. Fig. 2.The IU muscles unite in the midline to form a n arch over the deep dorsal vein. Just deep to the IU muscles, the deep dorsal vein of the penis bifurcates to follow the course of the dorsal arteries of the penis.
Fig. 3.A cross section of the midpoint of the ischiourethralis (IU) muscle illustrates the relationship of the IU to adjacent structures. The ischiocavernosi (IC) lie laterally, separated from the IU by septa of connective tissue. Note the thickness of the wall of the deep dorsal vein (ddv) in this location. CCP = corpora cavernosa penis; CS = corpus spongiosum. Six micrometer section stained with Gomori’s trichrome. x 43.
W.G IIAII, AN11 K.1). SACHS
Fig. 4. A representative cross section of the L, cord segment illustrating the location of neurons in the dorsolateral nucleus (DL)which were labelled by injection of the IU with FG.
Flg. 5. Neurons in the dorsolateral nucleus of spinal cord segment of L, labelled by FG injection of the IU. The arrows indicate processes which cross into the ventral white matter. Such processes probably
margin of the IU, the deep dorsal vein bifurcates to follow the two dorsal arteries of the penis. Injection of t h e retrograde tracer FG into the IU filled neurons in spinal cord segments L, and L,. In the three animals studied, neuronal counts were quite variable, ranging from 5 to 16 neurons (total cells in both segments). The majority of the labelled cells occurred in the L, segment in all animals. Most of the neurons were found in the dorsolateral nucleus (Figs. 4-6), although a n occasional cell was positioned more centrally in the gray matter of the ventral horn. In their copulation test, all males achieved intromission and, with the exception of one SHAM male, all ejaculated. There was no significant difference between groups on any measure of mating behavior. On the chief variable of intromission ratio, the mean values for both ejaculatory series were actually higher for IUx than SHAM males, although the differences did not approach significance (see Table 1). In the penile reflex test, one male in each group had no responses, and the groups were statistically indistinguishable on all measures. On key measures involving the number of responses for each class of erection,
represent axons while other processes which course medially or dorsally are dendrites. x 725. Flg. 6.A representative photomicrograph of a neuron to the IU. The labelled neuron is located centrally in the ventral gray matter (immediately dorsal to the dorsolateral nucleus). x 725.
TABLE 1. Mean (2SEM) values of intromission ratio SHAM
First ejaculation Second ejaculation
IUX males males ( n = 4) (n = 4) t'" P value 0.37 (0.12) 0.48 (0.12) 0.44 0.53 0.51 (0.14) 0.65 (0.08) 0.85 0.41
t.!. = value for students t test
P values were all >0.30. Among responders, all males displayed penile body lengthening, flips, Els, and E2s. All three SHAM responders displayed E3s, with individual values of 2, 4, and 10. Only one IUx male showed E3s, and he had six of these responses. DISCUSSION
These studies confirm that the arching band of tissue over the dorsal penile vein of the rat is a paired striated muscle. In all probability the muscle we have described is equivalent to the subpubic ligament of Greene ( 1968),the only previous reference to this tissue in the rat penis. Furthermore, considering the anatomy and
ISCHIOURETHI1A1,IS MUSCLE OF THE RAT
the pattern of innervation, it is likely that the muscle corresponds to the IU muscle described in other species (Houston, 1830; Christensen, 1954). An erectile function has been confirmed for this muscle in the dog (Hart, 1972; Colleen et al., 1981) and was implied by the anatomy of this muscle in the rat. However, no such function in the rat was discovered by our behavioral tests. In a re-examination of the “venae compressor dorsalis penis” muscle (of Houston, 1830) of the dog, Christensen (1954) noted that the muscle arose from the ischium and renamed it the ischiourethralis. Unlike other species in which the penile crura and the IU have separate origins from pelvic bones, the IU in the rat arises from the tunica albuginea of the crura and the penile bulb. In view of its attachments in the rat, the designation of the muscle we have described as the IU is somewhat confusing; perhaps, a reconsideration of the nomenclature is in order. The body of the IU in the rat is very closely apposed to the ischiocavernosus muscle. The affinity between the two muscles is reflected in the similar location of their motor neurons in the dorsolateral nucleus of the lumbar cord, which raises the possibility that these muscles may act in concert in the process of erection. The perineal muscles involved in sexual reflexes and the neurons which innervate them are under hormonal control (Breedlove and Arnold, 1980; Wee and Clemens, 1987). Consequently, the neurons in the male are more numerous and larger than in females (McKenna and Nadelhaft, 1986). Presumably, the sexual dimorphism would extend to the IU. A thin band of tissue dorsal to the clitoral vein was identified in rats, and electrical stimulation of this tissue produces weak contractions (Dail and Walton, unpublished observations). Although retrograde studies have not yet been done on this tissue, it is presumed to be homologous to the male’s IU. Removal of the IU did not prevent males from displaying normal copulatory behavior. The relatively high intromission ratio of the IUx males indicates that there was not a significant impairment in erection of the penile body, a requisite for intromission in rats (Sachs, 1983). However, because glans erection only occurs intravaginally during copulation in this species, results of reflex tests provide better evidence about the erectile function of the glans than measures of copulatory behavior. In the reflex test, IUx and SHAM males had a similar number of each class of erection of penile glans and body. It might be argued that other striated penile muscles compensated for the IU and provided adequate venous compression to permit erection. In a subsequent study, however, rats that had the BS, ischiocavernosus, and IU muscles excised were nonetheless able to display erections of the glans ( a t least E l s and E2s) and of the penile body (Sachs and Liu, unpublished observations). These results mean that the rat IU is not essential for erection of the penile body or glans, and the data reinforce earlier conclusions that substantial tumescence of the glans is possible by vascular means alone, without augmentation by the striated penile muscles (Sachs, 1982, 1983; Hart and Melese-d’Hospita1, 1983). Our finding also suggests that the severe disruption of copulatory behavior and reflexive erections consequent to motor pudendal nerve
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section (Sachs et al., 1989) was not likely to have been caused by denervation of the IU. Although this study establishes that the IU is not necessary for penile erection in rats, a contributory role for this muscle cannot yet be ruled out. The sample sizes were small, and larger samples might reveal a difference in the relative frequency of different erection intensities. Only one of the three responding IUx males displayed the highest intensity glans erections (cups), whereas all three SHAM responders had cups. Because the number of cups displayed by the responding IUx male was in the middle of the range displayed by the SHAM animals, the difference in response rate was not significant. It is not unusual for males to present no cups in a given reflex test, and therefore the number of males that might be needed to discover a role for the IU in promoting glans erections may be large. An alternative approach to the erectile role of the IU might be analogous to that adopted for analysis of the sexual function of the BS muscle (Sachs, 1982, 1983).Excision of this muscle (BSx) eliminates the display of penile cups in reflex tests, but the copulatory behavior of BSx males is normal, a s is the weight of the ejaculate and the total number of spermatozoa. However, in females mated to BSx males, the sperm count in the uterus is reduced and that in the vagina is increased. Presumedly, the reduced potential for forming cups results in a poor seal between glans and cervix, thereby reducing sperm transport into the uterus. A similar test might be best suited to discovering a physiological role for the IU in penile erection. ACKNOWLEDGMENTS The authors wish to express their gratitude to Guy Walton for anatomical studies, Denise B. McQuade for surgery and Yian-Cheng Liu for behavioral testing. The research was supported by grants ROlNS1983-07 and HD08933 from NIH and 0215-856 from The University of Connecticut Research Foundation. LITERATURE CITED Bitran, D., S.A. Miller, D.B. McQuade, R.E. Leipheimer, and B.D. Sachs 1988 Inhibition of sexual reflexes by lumbosacral injection of a GABAb agonist in male rats. Pharmacol. Biochem. Behav., 31.657-666. Breedlove, S.M., and A.P. Arnold 1980 Hormone accumulation in a sexually dimorphic motor nucleus of the rat spinal cord. Science, 21 0:564-566. Christensen, G.C. 1954 Angioarchitecture of the canine penis and the process of erection. Am. J. Anat., 95t227-261. Colleen, S., B. Holmquist, and T. Olin 1981 An angiographic study of erection in the dog. Urol. Res., 9t297-302. Greene, E.C. 1968 Anatomy of the Rat. Hafner Publishing Co., New York, p. 299. Hart. B.L. 1972 The action of extrinsic penile muscles during copulation in the male dog. Anat. Rec., 17c3t1-6. Hart, B.L., and R.L. Kitchell 1966 Penile erection and contraction of penile muscles in the spinal and intact dog Am. J. Physiol., 210. 257-262. Hart, B.L , and P.Y. Melese-d’Hospital 1983 Penile mechanisms and the role of the striated penile muscles in penile reflexes. Physiol. Behav., 31t807-813. Holmes, G.M., D.G. Holmes, and B.D. Sachs 1988 An IBM-PC based data collection system for recording rodent copulatory behavior and for general event recording. Physiol. Behav., 44t825-828 Houston, J . 1830 Compressor venae dorsalis penis. Dublin Hasp. Rep. and Comm. Med. Surg., 5:459-493. Leipheimer, R.E., and B.D. Sachs 1988 GABAergic regulation of penile reflexes and copulation in rats. Physiol. Behav., 42:351-357.
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McKenna, K., and I. Nadelhaft 1986 The organization of the pudendal nerve in the male and female rat. J. Comp. Neurol., 248:532-549. Purohit, R.C. and S.D. Beckett 1976 Penile pressures and muscle activity associated with erection and ejaculation in the dog. Am J. Physiol., 231t1343-1348. Rubin, E , and D. Purves 1980 Segmental organization of sympathetic preganglionic neurons in the mammalian spinal cord. J. Comp. Neurol., 193t163-174. Sachs, B.D. 1978 Conceptual and neural mechanisms of masculine copulatory behavior. In: Sex and Behavior: Status and Prospectus. T.E. McGill, D.A. Dewsbury, and B.D. Sachs, eds Plenum Press, New York, pp. 267-295. Sachs, B.D. 1982 Role of striated penile muscles in penile reflexes, copulation, and induction of pregnancy in the rat. J. Reprod Fertil. 66433-443. Saths, B.D. 1983 Potency and fertility: Hormonal and mechanical causes and effects of penile actions in rats. In: Hormones and
Behaviour in Higher Vertebrates. J. Balthazart, E. Prove, and R. Gilles, eds Springer-Verlag. Berlin, pp. 86-1 10. Sachs, B D., Y.C. Liu, D.B. McQuade, J.X. Wang, and G.H. Holmes 1989 Sensory and motor branches of pudendal nerve of male rats: Roles in copulation and reflexive erection. SOC. Neurosci. Abstr., 15t631. Schreder, H.D. 1980 Organization of the motoneurons innervating the pelvic muscles of the male rat. J. Comp. Neurol., 192.567-587. Sisson, S. 1975 Equine urogenital system. In. Sisson and Grossman’s, The Anatomy of Domestic Animals. R. Getty, ed. W.B. Saunders, Philadelphia, Vol. 1, pp. 524-549. Wallach, S.J.R., and B L. Hart 1983 The role of the striated penile muscles of the male rat in seminal plug dislodgement and deposition. Physiol. Behav., 31t815-821. Wee, B.E.F., and L.G. Clemens 1987 Characteristics of the spinal nucleus of the bulbocavernosus a r e influenced by genotype in the house mouse. Brain Res., 424.305-310.
The lschiourethralis Muscle of the Rat: Anatomy, Innervation, and Function WILLIAM G. DAIL AND BENJAMIN D. SACHS Department of Anatomy, University of New Mexico School of Medicine, Albuquerque, New Mexico (W.G.D.); and Department of Psychology, University of Connecticut (B.D.S.), Storrs, Connecticut
ABSTRACT The ischiourethralis (IU), a striated perineal muscle presumed to be involved in sexual reflexes, was studied in the rat. The paired muscle arises from the penile crus and the penile bulb and unites in a raphe over the deep dorsal vein of the penis. Retrograde tracing studies show that the muscle is innervated by neurons in the dorsolateral nucleus of the lumbar spinal cord, a pudendal nerve motor nucleus which also innervates the ischiocavernosus muscle. Excision of the IU muscle did not interfere with the ability of males to display normal copulatory behavior, nor did it affect significantly the number and intensity of reflexive erections. It nevertheless remains possible that the IU may contribute to intense glans erection by compressing the deep dorsal vein. The vascular events of penile erection are accompanied by patterned activity of striated penile muscles, including the ischiocavernosi (IC) and the bulbospongiosus (BS). The precise role of the extrinsic muscles may vary according to species and to the stage of copulatory behavior. For example, in addition to its role in the expulsion of semen, the BS muscle of dogs may force blood from the penile bulb to the glans during erection (Hart, 1972). A comparable action has been confirmed for the rat: the BS muscle augments vascular filling of the corpus spongiosum by compressing the penile bulb, and excision of this muscle eliminates full erection of the glans penis and reduces the fertility of mating (Sachs, 1982, 1983; Hart and Melese-d’Hospital, 1983). In the rat, the ischiocavernosus muscles are responsible for penile “flips,” which involve straightening the normal ventrof lexion of the penile body to permit intromission (Sachs, 1982, 1983; Hart and Melese-d’Hospita1, 1983). Similarly, the increased pressure of the corpora cavernosa penis of the dog during intromission is correlated with a n increase in the activity of the ischiocavernosus muscles (Purohit and Beckett, 1976). It has also been proposed that the paired ischiocavernosus muscles may aid in detumescence in the dog (Hart, 1972). Less is known of the role of a band of striated muscle which overlies the deep dorsal vein of the penis in some animals. Houston (1830) first described the muscle in the dog and other mammals and, because of its anatomical relationships, named it the venae compressor dorsalis penis muscle (also see Sisson, 1975). Christensen ( 1954), who renamed the muscle the ischiourethralis (IU), proposed that in the dog, the muscle lessened the free flow of blood through the deep dorsal vein after intromission. Later studies indicated that the IU in the dog contributes to rapid erection and is responsible for the tumescence of the glans penis (Hart and Kitchell, 1966; Hart, 1972). The rat is a frequently used species in which to investigate reproductive function and, while many studc
1991 WILEY-LISS, INC
ies have focused on the BS and the ischiocavernosus muscles (Schr@der,1980; Sachs, 1982, 1983; Hart and Melese-d’Hospita1, 1983; Wallach and Hart, 1983; McKenna and Nadelhaft, 19861, to our knowledge there have been no descriptive or functional studies of the IU in this important animal model. Little is known about the role of venous occlusion in penile erection in the rat, but the position of the IU overlying the deep dorsal penile vein suggests that this muscle may promote penile erection by reducing venous drainage. This hypothetical function is reinforced by the report (Sachs et al., 1989) that section of the motor branch of the pudendal nerve drastically impairs copulatory behavior and prevents rats from displaying even the vascular components of reflexive erection. Although the innervation of IU muscle in the rat remains to be determined, the pudendal nerve is a likely candidate, a s it innervates several other perineal striated muscles associated with sexual or eliminative functions (McKenna and Nadelhaft, 1986).The present investigation provides a n anatomical description of the muscle, identifies the spinal cord location of neurons which innervate it, and determines whether excision of the muscle impairs the mating behavior and erectile potential of rats. MATERIALS AND METHODS Anatomical Studies
The anatomical experiments were conducted on mature male (200 g) Sprague-Dawley rats. Dissection and histological studies were performed on the penes of seven rats which were fixed by perfusion with buffered 10% formalin preceded by flushing with phosphate-
Received May 21, 1990; accepted J u l y 24, 1990. Address reprint requests to William G. D a d , Department of Anatomy, University of New Mexico School of Medicine, Albuquerque, NM 87131.
buffered saline. Paraffin sections were taken through the IU at 6 pm and stained with Gomori’s trichrome to confirm the observations derived from the dissections. An additional three rats were used to determine the location and number of motor neurons which project to the IU. Rats were anesthetized with sodium pentobarbital (30 mgikg i.p.) and prepared for sterile surgery. The IU was exposed by a single incision a t the base of the penis, proximal to the suspensory ligament of the penis. To expose more of the IU, the adjacent ischiocavernosus muscle was partially removed. With the aid of a Picospritzer (General Valve Corp., Fairfield, N J ) , less than 1 pl of a suspension of 2.5% Fluorogold (FG; Flurochrome Inc., Englewood, CO) was injected into each side of the IU. Alternatively, a Hamilton syringe was used to deliver FG to the muscle. The needle tip, which entered the IU near its raphe, was directed laterally beneath the muscle epimysium. Injection of FG resulted in a yellow coloration of the muscle with no overt leakage of the dye. The entry point of the needle was sealed by a cautery. Seven days following the injection of the retrograde tracer, rats were anesthetized and perfused transcardially with phosphate-buffered saline containing procaine hydrochloride, followed by paraformaldehyde (4%) in phosphate buffer. Spinal cord sections L,-L,, identified by the criteria of Rubin and Purves (1980), were removed, rinsed of fixative, and stored overnight in 20% sucrose in phosphate buffer. The L,L, spinal cord segments of the rat contain the alpha motoneurons of the pudendal nerve including those supplying the ischiocavernosus and BS muscles (McKenna and Nadelhaft, 1986). Spinal cord segments were frozen, sectioned at 30 pm, and examined with a fluorescence microscope for labelled cells. A block of tissue containing the IU was also taken to verify the site of the injection of FG. Behavior Studies
Subjects Eight male rats (Long-Evans hooded, 120-150 days of age; 450-600 g) were selected on the basis of having copulated and displayed reflexive penile erections reliably in tests similar to those described below. The rats were randomly assigned to IU excision (IUx) or sham-operated (SHAM) groups ( n = 4 per group). Surgery
After anesthesia induced by i.m. injection of ketamine (50 mgikg) and xylazine ( 4 mgikg), the perigenital area was shaved, and a transverse, 1 cm incision was made in the skin just anterior to the palpable penile flexure. The penile body was then deflected ventrocaudally, and the suspensory ligament was removed to gain access to the IU. For IUx males, the paired muscle was carefully teased away from underlying tissue and cut near the lateral attachments as laterally as possible without disturbing adjacent tissue. Care was also taken to avoid damage to the dorsal penile nerves. For SHAM males, the IU was located but not otherwise disturbed. The incision in t h e skin was then closed with silk sutures, supplemented with a single 7 mm wound clip which was removed under ether anesthesia 6-7 days later. Surgery and recovery were uneventful in all animals.
Behavioral testing
Testing began 9-10 days after surgery. Details of the testing procedures, behavioral variables, and the computer software used for behavioral recording have been fully described elsewhere (Bitran e t al., 1988; Holmes e t al., 1988; Leipheimer and Sachs, 1988). Briefly, for the penile reflex test, a male was placed in supine position with its anterior torso restrained in a loosely fitting cylinder. The penile sheath was retracted to the base of the glans, and the glans and distal penile body were kept exposed by fastening a strip of paper tape, through which a hole had been punched, over the penis. No further stimulation was applied. Events recorded included penile body lengthening, straightening of the flexure (“flips”),and glans erection of three intensities classified as E l , E2, and E 3 or “cup.” Latencies were measured from sheath retraction, and tests continued for 15 minutes after the first reflex, or for 30 minutes if no responses occurred. Copulation was tested 1 week after the reflex test, during the dark phase of the 12:12 Light-Dark cycle in 40 x 21 x 25 cm glass chambers littered with wood shavings. Intromissions (I), mounts without intromission (M), and ejaculations were recorded and counted, and the latency to each behavior was measured from the time of placement of the hormone-induced estrous female into the cage with the male. A key measure of the erectile capacity of males is the intromission ratio (Sachs, 19781, defined as No. Ii(No. M + No. I). (Low intromission ratios can result from a large number of factors, including penile insensitivity, inappropriate orientation of mount, or lack of receptivity of the female. High intromission ratios require all of these and erection of the penile body so that the glans is directed rostrally instead of ventrally [Sachs, 1978, 1982, 19831.) Mating tests continued until the first intromission after the second ejaculation, or, if no ejaculation occurred. for 30 minutes after the first intromission. RESULTS The anatomy of the striated muscles of the r a t penis is depicted in Figure 1.The IC and the BS muscles have been described in detail by McKenna and Nadelhaft (1986) and will not be treated here. The IU is a paired muscle which forms an arch over the deep dorsal vein a t the base of the penile shaft (Fig. 2). The muscle is found a t the region where the pyramidalis muscle covers the pubic symphysis in the rat (Fig. 2). Dissection of the muscle laterally indicates t h a t i t has tendinous origins from the tunica albuginea of the penile crus and penile bulb. An origin from pubic bones was not apparent. Examination of histological sections revealed t h a t the IU is a striated muscle which overlies the dorsal penile arteries and dorsal penile nerves as well as the deep dorsal vein (Fig. 3). Although the IU is closely apposed to the ischiocavernosus, a connective tissue layer intervenes. A midline raphe was apparent in the gross specimens, but was less obvious in the histologic sections. Distally along the penile shaft, smooth muscle is a prominent component of the wall of the deep dorsal vein; however, the vein is relatively thin at the point where it underlies the IU (Fig. 3). At the proximal
205
ISCHIOUKETHRA1,IS MUSCLE OF THE RAT
pyramidalis muscle
...........pyramidalis muscle
...........muscle ischiocavernosus ..bulbospongiosus muscle
ischiourethralis muscle ischiocavernosus muscle deep dorsal vein
..rectum
-
‘ 1 = 3; -&-*.&>Y.g
1
..................shaft of penis
2
Fig. 1, The striated perineal muscles of the rat penis include the IC and the multilobed BS. The dorsal part of the BS passes behind the rectum. The region between the pyramidalis muscle and the base of the penis is shown in detail in Figure 2. Fig. 2.The IU muscles unite in the midline to form a n arch over the deep dorsal vein. Just deep to the IU muscles, the deep dorsal vein of the penis bifurcates to follow the course of the dorsal arteries of the penis.
Fig. 3.A cross section of the midpoint of the ischiourethralis (IU) muscle illustrates the relationship of the IU to adjacent structures. The ischiocavernosi (IC) lie laterally, separated from the IU by septa of connective tissue. Note the thickness of the wall of the deep dorsal vein (ddv) in this location. CCP = corpora cavernosa penis; CS = corpus spongiosum. Six micrometer section stained with Gomori’s trichrome. x 43.
W.G IIAII, AN11 K.1). SACHS
Fig. 4. A representative cross section of the L, cord segment illustrating the location of neurons in the dorsolateral nucleus (DL)which were labelled by injection of the IU with FG.
Flg. 5. Neurons in the dorsolateral nucleus of spinal cord segment of L, labelled by FG injection of the IU. The arrows indicate processes which cross into the ventral white matter. Such processes probably
margin of the IU, the deep dorsal vein bifurcates to follow the two dorsal arteries of the penis. Injection of t h e retrograde tracer FG into the IU filled neurons in spinal cord segments L, and L,. In the three animals studied, neuronal counts were quite variable, ranging from 5 to 16 neurons (total cells in both segments). The majority of the labelled cells occurred in the L, segment in all animals. Most of the neurons were found in the dorsolateral nucleus (Figs. 4-6), although a n occasional cell was positioned more centrally in the gray matter of the ventral horn. In their copulation test, all males achieved intromission and, with the exception of one SHAM male, all ejaculated. There was no significant difference between groups on any measure of mating behavior. On the chief variable of intromission ratio, the mean values for both ejaculatory series were actually higher for IUx than SHAM males, although the differences did not approach significance (see Table 1). In the penile reflex test, one male in each group had no responses, and the groups were statistically indistinguishable on all measures. On key measures involving the number of responses for each class of erection,
represent axons while other processes which course medially or dorsally are dendrites. x 725. Flg. 6.A representative photomicrograph of a neuron to the IU. The labelled neuron is located centrally in the ventral gray matter (immediately dorsal to the dorsolateral nucleus). x 725.
TABLE 1. Mean (2SEM) values of intromission ratio SHAM
First ejaculation Second ejaculation
IUX males males ( n = 4) (n = 4) t'" P value 0.37 (0.12) 0.48 (0.12) 0.44 0.53 0.51 (0.14) 0.65 (0.08) 0.85 0.41
t.!. = value for students t test
P values were all >0.30. Among responders, all males displayed penile body lengthening, flips, Els, and E2s. All three SHAM responders displayed E3s, with individual values of 2, 4, and 10. Only one IUx male showed E3s, and he had six of these responses. DISCUSSION
These studies confirm that the arching band of tissue over the dorsal penile vein of the rat is a paired striated muscle. In all probability the muscle we have described is equivalent to the subpubic ligament of Greene ( 1968),the only previous reference to this tissue in the rat penis. Furthermore, considering the anatomy and
ISCHIOURETHI1A1,IS MUSCLE OF THE RAT
the pattern of innervation, it is likely that the muscle corresponds to the IU muscle described in other species (Houston, 1830; Christensen, 1954). An erectile function has been confirmed for this muscle in the dog (Hart, 1972; Colleen et al., 1981) and was implied by the anatomy of this muscle in the rat. However, no such function in the rat was discovered by our behavioral tests. In a re-examination of the “venae compressor dorsalis penis” muscle (of Houston, 1830) of the dog, Christensen (1954) noted that the muscle arose from the ischium and renamed it the ischiourethralis. Unlike other species in which the penile crura and the IU have separate origins from pelvic bones, the IU in the rat arises from the tunica albuginea of the crura and the penile bulb. In view of its attachments in the rat, the designation of the muscle we have described as the IU is somewhat confusing; perhaps, a reconsideration of the nomenclature is in order. The body of the IU in the rat is very closely apposed to the ischiocavernosus muscle. The affinity between the two muscles is reflected in the similar location of their motor neurons in the dorsolateral nucleus of the lumbar cord, which raises the possibility that these muscles may act in concert in the process of erection. The perineal muscles involved in sexual reflexes and the neurons which innervate them are under hormonal control (Breedlove and Arnold, 1980; Wee and Clemens, 1987). Consequently, the neurons in the male are more numerous and larger than in females (McKenna and Nadelhaft, 1986). Presumably, the sexual dimorphism would extend to the IU. A thin band of tissue dorsal to the clitoral vein was identified in rats, and electrical stimulation of this tissue produces weak contractions (Dail and Walton, unpublished observations). Although retrograde studies have not yet been done on this tissue, it is presumed to be homologous to the male’s IU. Removal of the IU did not prevent males from displaying normal copulatory behavior. The relatively high intromission ratio of the IUx males indicates that there was not a significant impairment in erection of the penile body, a requisite for intromission in rats (Sachs, 1983). However, because glans erection only occurs intravaginally during copulation in this species, results of reflex tests provide better evidence about the erectile function of the glans than measures of copulatory behavior. In the reflex test, IUx and SHAM males had a similar number of each class of erection of penile glans and body. It might be argued that other striated penile muscles compensated for the IU and provided adequate venous compression to permit erection. In a subsequent study, however, rats that had the BS, ischiocavernosus, and IU muscles excised were nonetheless able to display erections of the glans ( a t least E l s and E2s) and of the penile body (Sachs and Liu, unpublished observations). These results mean that the rat IU is not essential for erection of the penile body or glans, and the data reinforce earlier conclusions that substantial tumescence of the glans is possible by vascular means alone, without augmentation by the striated penile muscles (Sachs, 1982, 1983; Hart and Melese-d’Hospita1, 1983). Our finding also suggests that the severe disruption of copulatory behavior and reflexive erections consequent to motor pudendal nerve
207
section (Sachs et al., 1989) was not likely to have been caused by denervation of the IU. Although this study establishes that the IU is not necessary for penile erection in rats, a contributory role for this muscle cannot yet be ruled out. The sample sizes were small, and larger samples might reveal a difference in the relative frequency of different erection intensities. Only one of the three responding IUx males displayed the highest intensity glans erections (cups), whereas all three SHAM responders had cups. Because the number of cups displayed by the responding IUx male was in the middle of the range displayed by the SHAM animals, the difference in response rate was not significant. It is not unusual for males to present no cups in a given reflex test, and therefore the number of males that might be needed to discover a role for the IU in promoting glans erections may be large. An alternative approach to the erectile role of the IU might be analogous to that adopted for analysis of the sexual function of the BS muscle (Sachs, 1982, 1983).Excision of this muscle (BSx) eliminates the display of penile cups in reflex tests, but the copulatory behavior of BSx males is normal, a s is the weight of the ejaculate and the total number of spermatozoa. However, in females mated to BSx males, the sperm count in the uterus is reduced and that in the vagina is increased. Presumedly, the reduced potential for forming cups results in a poor seal between glans and cervix, thereby reducing sperm transport into the uterus. A similar test might be best suited to discovering a physiological role for the IU in penile erection. ACKNOWLEDGMENTS The authors wish to express their gratitude to Guy Walton for anatomical studies, Denise B. McQuade for surgery and Yian-Cheng Liu for behavioral testing. The research was supported by grants ROlNS1983-07 and HD08933 from NIH and 0215-856 from The University of Connecticut Research Foundation. LITERATURE CITED Bitran, D., S.A. Miller, D.B. McQuade, R.E. Leipheimer, and B.D. Sachs 1988 Inhibition of sexual reflexes by lumbosacral injection of a GABAb agonist in male rats. Pharmacol. Biochem. Behav., 31.657-666. Breedlove, S.M., and A.P. Arnold 1980 Hormone accumulation in a sexually dimorphic motor nucleus of the rat spinal cord. Science, 21 0:564-566. Christensen, G.C. 1954 Angioarchitecture of the canine penis and the process of erection. Am. J. Anat., 95t227-261. Colleen, S., B. Holmquist, and T. Olin 1981 An angiographic study of erection in the dog. Urol. Res., 9t297-302. Greene, E.C. 1968 Anatomy of the Rat. Hafner Publishing Co., New York, p. 299. Hart. B.L. 1972 The action of extrinsic penile muscles during copulation in the male dog. Anat. Rec., 17c3t1-6. Hart, B.L., and R.L. Kitchell 1966 Penile erection and contraction of penile muscles in the spinal and intact dog Am. J. Physiol., 210. 257-262. Hart, B.L , and P.Y. Melese-d’Hospital 1983 Penile mechanisms and the role of the striated penile muscles in penile reflexes. Physiol. Behav., 31t807-813. Holmes, G.M., D.G. Holmes, and B.D. Sachs 1988 An IBM-PC based data collection system for recording rodent copulatory behavior and for general event recording. Physiol. Behav., 44t825-828 Houston, J . 1830 Compressor venae dorsalis penis. Dublin Hasp. Rep. and Comm. Med. Surg., 5:459-493. Leipheimer, R.E., and B.D. Sachs 1988 GABAergic regulation of penile reflexes and copulation in rats. Physiol. Behav., 42:351-357.
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McKenna, K., and I. Nadelhaft 1986 The organization of the pudendal nerve in the male and female rat. J. Comp. Neurol., 248:532-549. Purohit, R.C. and S.D. Beckett 1976 Penile pressures and muscle activity associated with erection and ejaculation in the dog. Am J. Physiol., 231t1343-1348. Rubin, E , and D. Purves 1980 Segmental organization of sympathetic preganglionic neurons in the mammalian spinal cord. J. Comp. Neurol., 193t163-174. Sachs, B.D. 1978 Conceptual and neural mechanisms of masculine copulatory behavior. In: Sex and Behavior: Status and Prospectus. T.E. McGill, D.A. Dewsbury, and B.D. Sachs, eds Plenum Press, New York, pp. 267-295. Sachs, B.D. 1982 Role of striated penile muscles in penile reflexes, copulation, and induction of pregnancy in the rat. J. Reprod Fertil. 66433-443. Saths, B.D. 1983 Potency and fertility: Hormonal and mechanical causes and effects of penile actions in rats. In: Hormones and
Behaviour in Higher Vertebrates. J. Balthazart, E. Prove, and R. Gilles, eds Springer-Verlag. Berlin, pp. 86-1 10. Sachs, B D., Y.C. Liu, D.B. McQuade, J.X. Wang, and G.H. Holmes 1989 Sensory and motor branches of pudendal nerve of male rats: Roles in copulation and reflexive erection. SOC. Neurosci. Abstr., 15t631. Schreder, H.D. 1980 Organization of the motoneurons innervating the pelvic muscles of the male rat. J. Comp. Neurol., 192.567-587. Sisson, S. 1975 Equine urogenital system. In. Sisson and Grossman’s, The Anatomy of Domestic Animals. R. Getty, ed. W.B. Saunders, Philadelphia, Vol. 1, pp. 524-549. Wallach, S.J.R., and B L. Hart 1983 The role of the striated penile muscles of the male rat in seminal plug dislodgement and deposition. Physiol. Behav., 31t815-821. Wee, B.E.F., and L.G. Clemens 1987 Characteristics of the spinal nucleus of the bulbocavernosus a r e influenced by genotype in the house mouse. Brain Res., 424.305-310.
