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Differential effect of castration on the somal size of pudendal motoneurons in the adult male rat
326
Brain Research, 577 (1992) 326-330 Elsevier
BRES 25146
Differential effect of castration on the somal size of pudendal motoneurons in the adult male rat William E Collins III, Andrew W. Seymour* and Sharon W. Klugewicz Department of Neurobiology and Behavior, State University of New York at Stony Brook, Stony Brook, NY 11794-5230 (USA)
(Accepted 21 January 1992) Key words: Androgen; Penis; Sexual dimorphism; Sexual reflex; Spinal cord
We have examined the organization of the androgen sensitivity of pudendal motoneurons in adult male rats. Comparisons of mean somal cross sectional area of pudendal motoneurons identified by retrograde labeling with horseradish peroxidase were made between three treatment groups: castrates with testosterone propionate supplementation, castrates with placebo, and sham castrates. Bulbospongiosus and ischiocavernosus motoneurons were sensitive to androgen treatment, whereas external anal sphincter and external urethral sphincter motoneurons were not. The somatic c o m p o n e n t of the p u d e n d a l m o t o r system in male rats consists of the bulbospongiosus, ischiocavernosus, external anal sphincter, and external urethral sphincter muscles and motoneurons. The pudendal motoneurons have been localized to the dorsomedial (DM) and dorsolateral (DL) nuclei in the fifth and sixth lumbar segments of the spinal cord 2'18'25"3°. The pudendal m o t o r system is clearly involved in sexual function. The bulbospongiosus and ischiocavernosus muscles are attached to the penis 1°m'ls'22 and are active during copulation t°'22. Bulbospongiosus and ischiocavernosus motoneurons and muscles are sexually dimorphic in that they are present in adult males but vestigial in adult females 2. This sexual dimorphism results from the high levels of androgens occurring neonatally in males which prevents the involution of bulbospongiosus and ischiocavernosus muscles and the p r o g r a m m e d death of bulbospongiosus and ischiocavernosus m o t o n e u r o n s 25"26. The rat p u d e n d a l m o t o r system also exhibits androgen d e p e n d e n t plasticity in adulthood. Castration results in a m a r k e d attenuation of penile flips and penile cup formation in ex copula tests which is reversed by administration of exogenous testosterone or 5a-dihydrotestosterone 8'9. Mean somal cross sectional area of D M motoneurons is smaller in castrated males c o m p a r e d to gonadally intact males or castrated males treated with testosterone 3'7A3'16'17. Also, a 50% decrease in mean dendritic length of bulbospongiosus m o t o n e u r o n s retrogradely labeled with cholera toxin conjugated to horseradish peroxidase following castration has been re-
p o r t e d 7'~3, although a significant castration induced change in dendritic length was not observed in bulbospongiosus m o t o n e u r o n s intracellularly filled with horseradish peroxidase ( H R P ) 24. In addition, recent electron microscopic studies of bulbospongiosus m o t o n e u r o n somata and proximal dendrites have revealed that the percent surface area covered by synaptic boutons a5'~6 and the length of gap junctions 17 decrease following castration. The present study was u n d e r t a k e n to examine the organization of androgen sensitivity among p u d e n d a l motoneurons in adult male rats. While it is well documented that bulbospongiosus m o t o n e u r o n somal size is decreased in androgen deprived rats, it is not known whether other p u d e n d a l m o t o n e u r o n s exhibit this same androgen dependence. To resolve this question, we have used retrograde transport of H R P to identify bulbospongiosus, ischiocavernosus, external anal sphincter and external urethral sphincter m o t o n e u r o n s and measured the m o t o n e u r o n somal cross sectional areas under different treatment conditions. All surgical procedures were p e r f o r m e d aseptically under deep anesthesia using a mixture of K e t a m i n e and Xylazine (90 and 10 mg/kg i.m., resp.) with supplemental doses given as required to maintain areflexia (lack of withdrawal following pinching of the forepaw). Male S p r a g u e - D a w l e y rats were obtained at 60 days of age and either castrated or sham castrated (Sham). In addition, each castrated rat received a 30-day slow release pellet of either testosterone propionate (in a biodegradable matrix) ( C + T P ) or placebo (biodegradable matrix
* Present address: Department of Pathology, SUNY at Brooklyn, Health Science Center, Brooklyn, NY 11203, USA. Correspondence: W.E Collins III, Department of Neurobiology and Behavior. SUNY at Stony Brook. Stony Brook, NY 11794-5230, USA.
327 TABLE I Summary o f HRP injections
aNumber of rats; range of number of labeled motoneurons per rat,
Injected muscle VBS DBS EAS IC EUS
Sham a
C+ TP ~
C+ P~
5; 3; 7; 6; 4;
4; 4; 5; 6; 5;
6; 5; 6; 9; 6;
2-57 7-30 4-24 19-30 25-34
9-69 3-10 3-26 8-51 16-66
15-59 5-32 4-14 5-31 24-80
alone) (C+P) which was inserted subcutaneously into the scrotum. The biodegradable matrix consisted of cholesterol, microcrystaline cellulose, a-lactose, di- and tricalcium phosphate, calcium and magnesium stearate, and stearic acid. The pellets were obtained commercially from Innovative Research of America (Toledo, OH). Twenty-eight days following the initial surgery, the rats were weighed and reanesthetized. The perineal musculature was surgically exposed using a midline ventral incision. A single, discrete injection of H R P (Sigma, type VI; 0.5-1.0 kd; 50% in distilled H 2 0 ) was made into either the ventral bulbospongiosus (or bulbocavernosus), dorsal bulbospongiosus (or levator ani), ischiocavernosus, or external urethral sphincter muscles. In the case of ventral bulbospongiosus muscle injections, no distinction was made between medial and lateral portions of the muscle. Injection of the external anal sphincter muscle differed in that multiple (maximum of 4) injections were made through the perianal skin. In the majority of the experiments, several perineal muscles (maximum of 3) were injected with H R P in the same rat. Only muscles innervated by different motor nuclei were injected with H R P in any given rat, and, in most cases, sexually dimorphic and non-sexually dimorphic muscles were injected in the same rat. Table I provides a summary of the number of rats used and the number of labeled motoneurons for each muscle injection. Following an additional survival period of 48 h, each rat was deeply reanesthetized and transcardially peffused with 300-400 ml of saline containing lidocaine (0.005%) and heparin (500 units) at 37°C followed by 1 liter of perfusate consisting of a mixture of 1% paraformaldehyde and 1.25% glutaraldehyde in 0.1 M phosphate buffer (PB; pH 7.4) at 4°C. The L3-S 2 spinal cord was removed and blocked to distinguish left from right in the longitudinal plane of section. The penis along with attached musculature (bulbospongiosus and ischiocavernosus muscles) was removed and weighed. The excised spinal cords were postfixed in perfusate at 4°C for 2 h and then placed in 30% sucrose in 0.1 M PB at 4°C for
at least 12 h. Horizontal longitudinal sections (50/~m) were cut on a freezing microtome. The sections were processed using a tetramethyl benzidine method 19 with a glucose oxidase modification 12, counterstained with Neutral red, dehydrated, cleared in xylene and coverslipped in Permount. Without knowledge of the treatment subgroup, the spinal cord sections were examined light microscopically. Each HRP-labeled profile containing a nucleus (see Table I for numbers) was focused at the level of the nucleus and the perimeter was traced using a drawing tube attachment (x 500 magnification). These traces were then entered into a computerized cell measuring system by retracing the drawings with a digitizing tablet, and somal cross sectional area was computed. No correction was made for tissue shrinkage. In all cases, HRP-labeled motoneurons were observed only in DM or DL nuclei ipsilateral to the site of injection. In agreement with earlier studies 18'3°, labeled bulbospongiosus (dorsal and ventral) and external anal sphincter motoneurons were located throughout the DM nucleus, and labeled ischiocavernosus and external urethral sphincter motoneurons were restricted to the medial and lateral subdivisions, respectively, of the DL nucleus. In Sham experiments, a nested analysis of variance (ANOVA) consisting of the five populations of pudendal motoneurons (groups), the rats in each group (subgroups), and the observations from each rat indicated significant differences in mean somal cross sectional area between pudendal motoneurons innervating different muscles (P < 0.05). Subsequent pairwise post hoc comparisons (two tailed t-test) revealed that mean somal cross sectional areas of external anal sphincter and external urethral sphincter motoneurons were significantly smaller (P < 0.01) than those of bulbospongiosus and ischiocavernosus motoneurons, respectively. No significant difference was seen between mean somal cross sectional areas of motoneurons innervating bulbospongiosus and ischiocavernosus motoneurons or between external anal sphincter and external urethral sphincter motoneurons.
TABLE II Summary o f treatment effects on body weight and weight o f penis and attached muscles
Unweighted mean _+ S.E.M. aWeight of penis and attached muscles expressed as a percentage of body weight. Treatment
No. o f rats
Body weight (g)
Penis and muscle a
Sham C+TP C+P
12 13 17
402.1 + 10.3 392.7 + 7.8 380.2 ___6.9
0.43 + 0.01 0.45 + 0.03 0.20 + 0.01"
*P < 0.01; Sham vs. C+P comparison.
328 TABLE III Summary o f statistical analyses o f treatment effects on pudendal motoneuron somal cross sectional area
a See legend of Fig. 1 for explanation of abbreviations; b see text for formula; c degrees of freedom among subgroups from nested ANOVA see text; d established using Dunnet's test (two-tailed; ref. 5); n.s., not significant. Planned comparisons o f means
Motoneuron VBS DBS EAS IC EUS
Sham vs. C + T P
Sham vs. C + P
? (drY)
t b (df;e d)
1.195 1.831 1.119 0.623 0.450
2.893 2.723 0.164 3.809 0.762
(12; n.s.) ( 9; n.s.) (15; n.s.) (18; n.s.) (12; n.s.)
(12; ( 9; (15; (18; (12;
0.05) 0.05) n.s.) 0.01) n.s.)
Total body weight and the weight of the penis and attached muscles were analyzed to assess the effectiveness of the testosterone treatment. No significant difference in total b o d y weight was detected across t r e a t m e n t subgroups (Table II). Penis and attached muscle weight (expressed as percent of total body weight) was 50% smaller in the C + P subgroup c o m p a r e d to the sham subgroup ( P < 0.01; two tailed t-test), whereas, the sham and C + T P subgroups were statistically the same (Table II). Thus, the castration effectively r e m o v e d endogenous testosterone, and the castration induced atrophy of the penis and attached muscles was p r e v e n t e d by the testosterone pellet implants.
Somal
Cross Sectional
800700
L ..-.--
~I
Area Sham C+TP C+P
o
I VBS DBS EAS
IC
EUS
Fig. 1. A bar chart of mean somal cross sectional areas of ventral bulbospongiosus (VBS), dorsal bulbospongiosus (DBS), external anal sphincter (EAS), ischiocavernosus (IC), and external urethral sphincter (EUS) motoneurons in each treatment group is presented. The means are unweighted means, and the standard error bars were calculated using a random effects model. Significant testosterone treatment effects were detected in the cases of VBS, DBS, and IC motoneurons (Sham vs. C+P comparisons; ~P < 0.01; 2p < 0.05). See text for details concerning statistical tests.
Based on previously published observations 2'3'7n3't6, was anticipated that significant differences in motoneuron somal cross sectional areas may be detected between the sham and C + P treatment groups but not between the sham and C + T P t r e a t m e n t groups. Therefore, two planned comparisons, sham vs. C + P and sham vs. C + T P , were carried out on means in each of the five populations of m o t o n e u r o n s using a Student's t-test with the following formula28:
17 it
where m i and ni are the unweighted mean and n u m b e r respectively of all the observations within a t r e a t m e n t group, and MSsubgroups is the mean square among subgroups. D u n n e t t ' s test 5 for comparing two treatments to one control was done, and the degrees of f r e e d o m associated with the MSsubgroup values (•(ai-1); where a i is the number of rats in each treatment group) were used to determine significance (see Table III). Because there was significant variability between animals within treatments, a nested A N O V A consisting of t r e a t m e n t groups, rats in each t r e a t m e n t group (subgroups), and somal cross sectional area measurements o b t a i n e d from each rat was done for each population of m o t o n e u r o n s to estimate MSsubgroups, between rat variability, and within rat variability. The between rat variability and within rat variability were used to obtain an unbiased estimate of the variance of an observation, and this estimate was in turn used to obtain the estimate of the standard error of each mean. As can be seen in Fig. 1 and Table III, no difference in somal cross sectional area between the C + T P and sham t r e a t m e n t groups was detected within any of the populations of motoneurons. Significant decreases (1520%) in mean somal cross sectional areas were observed in the C + P c o m p a r e d to the sham treatment groups in the cases of dorsal and ventral bulbospongiosus (P < 0.05) and ischiocavernosus ( P < 0.01) motoneurons. In contrast, no treatment effect on m e a n somal size was detected for external anal sphincter and external urethral sphincter motoneurons. Thus, testosterone has a differential effect on somal size among p u d e n d a l m o t o n e u r o n s in adult male rats. The sexually dimorphic bulbospongiosus and ischiocavernosus m o t o n e u r o n s were sensitive to androgen treatment, whereas the non-sexually dimorphic external anal sphincter and external urethral sphincter m o t o n e u r o n s were not. The observed androgen dependence of bulbospongiosus m o t o n e u r o n somal size was expected and agrees with previously r e p o r t e d studies TM ~5-17. In the case of ischiocavernosus m o t o n e u r o n somal size, a similar d e p e n d e n c e upon androgen in a d u l t h o o d
329 was suspected, since, like bulbospongiosus m o t o n e u r o n s , ischiocavernosus m o t o n e u r o n s are sexually dimorphic. The lack of androgen sensitivity in the case of external anal sphincter m o t o n e u r o n s has b e e n previously suggested based u p o n indirect evidence obtained from testosterone-treated neonatal female rats 14. However, this is the first direct study of possible androgen effects on rat external anal sphincter and external urethral sphincter
rons throughout the spinal cord and brainstem accumulate androgen 23, and external anal sphincter and external urethral sphincter m o t o n e u r o n s do not exhibit this androgen dependence. F u r t h e r m o r e , the bulbospongiosus muscles accumulate androgen 29 and exhibit marked atrophy following castration 6'31'32. R a n d and Breedlove have reported that unilateral local application of testosterone to bulbospongiosus muscles in castrated adult
m o t o n e u r o n s in adulthood. The results of this study have important consequences for future experiments. It is clear that the D M and D L
male rats produces differential effects on D M m o t o n e u ron dendritic length and arbor shape 2°'21. Also, Araki et
m o t o n e u r o n s are heterogeneous not only with respect to target musculature but also with respect to androgen
toneurons axotomized and allowed to innervate grafted soleus muscle is no longer testosterone d e p e n d e n t ,
sensitivity in adulthood. Therefore, it is essential to positively identify individual populations of D M and D L
whereas D M m o t o n e u r o n s axotomized and allowed to reinnervate perineal muscles retain androgen sensitivity.
m o t o n e u r o n s when undertaking steroid m a n i p u l a t i o n studies. F r o m a practical perspective, external anal
Thus, the observed effect of testosterone deprivation on bulbospongiosus, as well as ischiocavernosus, motoneu-
sphincter or external urethral sphincter m o t o n e u r o n s can be used as a control population of m o t o n e u r o n s to differentiate between general effects of androgens on mo-
ron somal size may be secondary to atrophy of the target musculature as opposed to a direct effect at the level of the m o t o n e u r o n s .
toneurons and effects specific to 'penile m o t o n e u r o n s ' . These results also address the question of the site of action of testosterone in the m a i n t e n a n c e of bulbospongiosus and ischiocavernosus m o t o n e u r o n somal size. It is
al.1 have demonstrated that m e a n somal size of D M mo-
spongiosus m o t o n e u r o n s in adult male rats because autoradiographic studies indicate that m o t o n e u r o n s in the D M nuclei accumulate androgens 2'4. However, m o t o n e u -
We would like to thank L.M. Mendell for the use of his facilities; D. Malysz for technical assistance; N.R. Mendell for help with the statistical methodology; and K.R. Peshori for reading a previous version of the manuscript. This work was supported by R01NS24206 and BNS-9111207 (W.EC.) and a summer fellowship from the Undergraduate Research and Creative Activities Program, SUNY at Stony Brook (S.W.K.). Some assistance was also provided by P01-NS14899 and R01-NSI6996 to L.M. Mendell.
1 Araki, I., Harada, Y. and Kuno, M., Target-dependent hormonal control of neuron size in the rat spinal nucleus of the bulbocavernosus, J. Neurosci., 11 (1991) 3025-3033. 2 Breedlove, B.S. and Arnold, A.P., Hormone accumulation in a sexually dimorphic motor nucleus of the rat spinal cord, Science, 210 (1980) 564-566. 3 Breedlove, B.S. and Arnold, A.P., Sexually dimorphic motor nucleus in the rat spinal cord: response to adult hormone manipulation, absence in androgen insensitive rats, Brain Res., 225 (1981) 297-307. 4 Breedlove, B.S. and Arnold, A.P., Sex differences in the pattern of steroid accumulation by motoneurons of the rat lumbar spinal cord, J. Comp. Neurol., 215 (1983) 211-216. 5 Dunnett, C.W., New tables for multiple comparisons with a control, Biometrics, 82 (1964) 482-491. 6 Fukuda, Y., Honma, M., Saito, T., Tsunenari, Y. and Maekawa, K., Levator ani muscle and internal secretion in neonatally castrated rats, J. Tokyo Med. Col., 28b (1970) 815-819. 7 Goldstein, L.A., Kurz, E.M. and Sengelaub, D.R., Androgen regulation of dendritic growth and retraction in the development of a sexually dimorphic spinal nucleus, J. Neurosci., 10 (1990) 935-946. 8 Hart, B.L., Testosterone regulation of penile reflexes in male rats, Science, 155 (1967) 1283-1284. 9 Hart, B.L., Activation of sexual reflexes of male rats by dihydrotestosterone but not estrogen, PhysioL Behav., 23 (1979) 107-109. 10 Hart, B.L. and Melese-d'Hospital, P.Y., Penile mechanisms and the role of the striated penile muscles in penile reflexes, Physiol. Behav., 31 (1983) 807-813. 11 Hayes, K.J., The so-called 'levator ani' of the rat, Acta. Endo-
crinol., 48 (1965) 337-347. 12 Itoh, K., Konishi, A., Nomura, S., Mizuno, N., Nakamura, Y. and Sugimoto, T., Application of coupled oxidation reaction to electron microscopic demonstration of horseradish peroxidase: colbalt-glucose oxidase method, Brain Res., 175 (1979) 341346. 13 Kurz, E.M., Sengelaub, D.R. and Arnold, A.P., Androgens regulate the dendritic length of mammalian motoneurons in adulthood, Science, 232 (1986) 395-398. 14 Lee, J.H., Jordan, C.L. and Arnold, A.P., Critical period for androgenic regulation of soma size of sexually dimorphic motoneurons in rat lumbar spinal cord, Neurosci. Lett., 98 (1989) 79-84. 15 Leedy, M.G., Beattie, M.S. and Bresnahan, J.C., Testosteroneinduced plasticity of synaptic inputs to adult mammalian motoneurons, Brain Res., 424 (1987) 386-390. 16 Matsumoto, A., Micevych, P.E. and Arnold, A.P., Androgen regulates synaptic input to motoneurons of the adult rat spinal cord, J. Neurosci., 8 (1988) 4168-4176. 17 Matsumoto, A., Arnold, A.P., Zampighi, G.A. and Micevych, P.E., Androgenic regulation of gap junctions between motoneurons in the rat spinal cord, J. Neurosci., 8 (1988) 4177-4183. 18 McKenna, K.E. and Nadelhaft, I., The organization of the pudendal nerve in the male and female rat, J. Comp. Neurol., 248 (1986) 532-549. 19 Mesulam, M.-M., Tetramethyl benzidine for horseradish peroxidase neurohistochemistry: a non-carcinogenic blue reactionproduct with superior sensitivity for visualizing afferents and efferents, J. Histochem. Cytochem., 26 (1978) 106-117. 20 Rand, M.N. and Breedlove, S.M., Unilateral administration of androgen to bulbocavernosus and subsequent effects on SNB
generally assumed that androgens act directly on bulbo-
330 dendritic length, Soc. Neurosci. Abstr., 15 (1989) 377. 21 Rand, M.N., Lofthus, B.E and Breedlove, S.M., Differential retraction of SNB dendritic arbors with unilateral testosterone treatment of BC/LA muscles, Soc. Neurosci. Abstr., 17 (1991) 535. 22 Sachs, B.C., Role of penile muscles in penile reflexes, copulation and induction of pregnancy in the rat, J. Reprod. Fertil., 66 (1982) 433-443. 23 Sar, M. and Stumpf, W.E., Androgen concentration in motor neurons of cranial nerves and spinal cord, Science, 197 (1977) 77-79. 24 Sasaki, M. and Arnold, A.P., Androgenic regulation of dendritic trees of motoneurons in the spinal nucleus of the bulbocavernosus: reconstruction after intracellular iontophoresis of horseradish peroxidase, J. Cornp. Neurol., 308 (1991) 11-27. 25 Schroder, H.D., Organization of motoneurons innervating the pelvic muscles of the male rat, J. Comp. Neurol., 192 (1980) 567-587. 26 Sengelaub, D.R. and Arnold, A.P., Hormonal control of neuron number in sexually dimorphic spinal nuclei of the rat: I. Testosterone-regulated death in the dorsolateral nucleus, J.
Comp. Neurol., 28 (1989) 622-629. 27 Sengelaub, D.R., Jordan, C.L., Kurz, E.M. and Arnold, A.P., Hormonal control of neuron number in sexually dimorphic spinal nuclei of the rat: II. Development of the spinal nucleus of the bulbocavernosus in androgen-insensitive (Tfm) rats, J. Comp. Neurol., 280 (1989) 630-636. 28 Sokal, R.R. and Rolhf, EJ., Biometry, 2nd edn., W.H. Freeman, San Fransisco, 1981, 243 pp. 29 Tremblay, R.R., Dube, J.Y., Ho-kim, M.A. and Lesage, R., Determination of rat muscle androgen complexes with methyltrienolone, Steroids, 29 (1977) 185-195. 30 Ueyama, T., Arakawa, H. and Mizuno, N., Central distribution of efferent and afferent components of the pudendal nerve in rat, Anat. Ernbryol., 177 (1987) 37-49. 31 Venable, J.H., Morphology of the cells of normal, testosterone deprived and testosterone-stimulated levator ani muscles, Am. J. Anat,, 119 (1966) 271-302. 32 Wainman, P. and Shipounoff, G.G., The effects of castration and testosterone propionate on the striated perineal musculature in the rat, Endocrinology, 29 (1941) 975-978.
Brain Research, 577 (1992) 326-330 Elsevier
BRES 25146
Differential effect of castration on the somal size of pudendal motoneurons in the adult male rat William E Collins III, Andrew W. Seymour* and Sharon W. Klugewicz Department of Neurobiology and Behavior, State University of New York at Stony Brook, Stony Brook, NY 11794-5230 (USA)
(Accepted 21 January 1992) Key words: Androgen; Penis; Sexual dimorphism; Sexual reflex; Spinal cord
We have examined the organization of the androgen sensitivity of pudendal motoneurons in adult male rats. Comparisons of mean somal cross sectional area of pudendal motoneurons identified by retrograde labeling with horseradish peroxidase were made between three treatment groups: castrates with testosterone propionate supplementation, castrates with placebo, and sham castrates. Bulbospongiosus and ischiocavernosus motoneurons were sensitive to androgen treatment, whereas external anal sphincter and external urethral sphincter motoneurons were not. The somatic c o m p o n e n t of the p u d e n d a l m o t o r system in male rats consists of the bulbospongiosus, ischiocavernosus, external anal sphincter, and external urethral sphincter muscles and motoneurons. The pudendal motoneurons have been localized to the dorsomedial (DM) and dorsolateral (DL) nuclei in the fifth and sixth lumbar segments of the spinal cord 2'18'25"3°. The pudendal m o t o r system is clearly involved in sexual function. The bulbospongiosus and ischiocavernosus muscles are attached to the penis 1°m'ls'22 and are active during copulation t°'22. Bulbospongiosus and ischiocavernosus motoneurons and muscles are sexually dimorphic in that they are present in adult males but vestigial in adult females 2. This sexual dimorphism results from the high levels of androgens occurring neonatally in males which prevents the involution of bulbospongiosus and ischiocavernosus muscles and the p r o g r a m m e d death of bulbospongiosus and ischiocavernosus m o t o n e u r o n s 25"26. The rat p u d e n d a l m o t o r system also exhibits androgen d e p e n d e n t plasticity in adulthood. Castration results in a m a r k e d attenuation of penile flips and penile cup formation in ex copula tests which is reversed by administration of exogenous testosterone or 5a-dihydrotestosterone 8'9. Mean somal cross sectional area of D M motoneurons is smaller in castrated males c o m p a r e d to gonadally intact males or castrated males treated with testosterone 3'7A3'16'17. Also, a 50% decrease in mean dendritic length of bulbospongiosus m o t o n e u r o n s retrogradely labeled with cholera toxin conjugated to horseradish peroxidase following castration has been re-
p o r t e d 7'~3, although a significant castration induced change in dendritic length was not observed in bulbospongiosus m o t o n e u r o n s intracellularly filled with horseradish peroxidase ( H R P ) 24. In addition, recent electron microscopic studies of bulbospongiosus m o t o n e u r o n somata and proximal dendrites have revealed that the percent surface area covered by synaptic boutons a5'~6 and the length of gap junctions 17 decrease following castration. The present study was u n d e r t a k e n to examine the organization of androgen sensitivity among p u d e n d a l motoneurons in adult male rats. While it is well documented that bulbospongiosus m o t o n e u r o n somal size is decreased in androgen deprived rats, it is not known whether other p u d e n d a l m o t o n e u r o n s exhibit this same androgen dependence. To resolve this question, we have used retrograde transport of H R P to identify bulbospongiosus, ischiocavernosus, external anal sphincter and external urethral sphincter m o t o n e u r o n s and measured the m o t o n e u r o n somal cross sectional areas under different treatment conditions. All surgical procedures were p e r f o r m e d aseptically under deep anesthesia using a mixture of K e t a m i n e and Xylazine (90 and 10 mg/kg i.m., resp.) with supplemental doses given as required to maintain areflexia (lack of withdrawal following pinching of the forepaw). Male S p r a g u e - D a w l e y rats were obtained at 60 days of age and either castrated or sham castrated (Sham). In addition, each castrated rat received a 30-day slow release pellet of either testosterone propionate (in a biodegradable matrix) ( C + T P ) or placebo (biodegradable matrix
* Present address: Department of Pathology, SUNY at Brooklyn, Health Science Center, Brooklyn, NY 11203, USA. Correspondence: W.E Collins III, Department of Neurobiology and Behavior. SUNY at Stony Brook. Stony Brook, NY 11794-5230, USA.
327 TABLE I Summary o f HRP injections
aNumber of rats; range of number of labeled motoneurons per rat,
Injected muscle VBS DBS EAS IC EUS
Sham a
C+ TP ~
C+ P~
5; 3; 7; 6; 4;
4; 4; 5; 6; 5;
6; 5; 6; 9; 6;
2-57 7-30 4-24 19-30 25-34
9-69 3-10 3-26 8-51 16-66
15-59 5-32 4-14 5-31 24-80
alone) (C+P) which was inserted subcutaneously into the scrotum. The biodegradable matrix consisted of cholesterol, microcrystaline cellulose, a-lactose, di- and tricalcium phosphate, calcium and magnesium stearate, and stearic acid. The pellets were obtained commercially from Innovative Research of America (Toledo, OH). Twenty-eight days following the initial surgery, the rats were weighed and reanesthetized. The perineal musculature was surgically exposed using a midline ventral incision. A single, discrete injection of H R P (Sigma, type VI; 0.5-1.0 kd; 50% in distilled H 2 0 ) was made into either the ventral bulbospongiosus (or bulbocavernosus), dorsal bulbospongiosus (or levator ani), ischiocavernosus, or external urethral sphincter muscles. In the case of ventral bulbospongiosus muscle injections, no distinction was made between medial and lateral portions of the muscle. Injection of the external anal sphincter muscle differed in that multiple (maximum of 4) injections were made through the perianal skin. In the majority of the experiments, several perineal muscles (maximum of 3) were injected with H R P in the same rat. Only muscles innervated by different motor nuclei were injected with H R P in any given rat, and, in most cases, sexually dimorphic and non-sexually dimorphic muscles were injected in the same rat. Table I provides a summary of the number of rats used and the number of labeled motoneurons for each muscle injection. Following an additional survival period of 48 h, each rat was deeply reanesthetized and transcardially peffused with 300-400 ml of saline containing lidocaine (0.005%) and heparin (500 units) at 37°C followed by 1 liter of perfusate consisting of a mixture of 1% paraformaldehyde and 1.25% glutaraldehyde in 0.1 M phosphate buffer (PB; pH 7.4) at 4°C. The L3-S 2 spinal cord was removed and blocked to distinguish left from right in the longitudinal plane of section. The penis along with attached musculature (bulbospongiosus and ischiocavernosus muscles) was removed and weighed. The excised spinal cords were postfixed in perfusate at 4°C for 2 h and then placed in 30% sucrose in 0.1 M PB at 4°C for
at least 12 h. Horizontal longitudinal sections (50/~m) were cut on a freezing microtome. The sections were processed using a tetramethyl benzidine method 19 with a glucose oxidase modification 12, counterstained with Neutral red, dehydrated, cleared in xylene and coverslipped in Permount. Without knowledge of the treatment subgroup, the spinal cord sections were examined light microscopically. Each HRP-labeled profile containing a nucleus (see Table I for numbers) was focused at the level of the nucleus and the perimeter was traced using a drawing tube attachment (x 500 magnification). These traces were then entered into a computerized cell measuring system by retracing the drawings with a digitizing tablet, and somal cross sectional area was computed. No correction was made for tissue shrinkage. In all cases, HRP-labeled motoneurons were observed only in DM or DL nuclei ipsilateral to the site of injection. In agreement with earlier studies 18'3°, labeled bulbospongiosus (dorsal and ventral) and external anal sphincter motoneurons were located throughout the DM nucleus, and labeled ischiocavernosus and external urethral sphincter motoneurons were restricted to the medial and lateral subdivisions, respectively, of the DL nucleus. In Sham experiments, a nested analysis of variance (ANOVA) consisting of the five populations of pudendal motoneurons (groups), the rats in each group (subgroups), and the observations from each rat indicated significant differences in mean somal cross sectional area between pudendal motoneurons innervating different muscles (P < 0.05). Subsequent pairwise post hoc comparisons (two tailed t-test) revealed that mean somal cross sectional areas of external anal sphincter and external urethral sphincter motoneurons were significantly smaller (P < 0.01) than those of bulbospongiosus and ischiocavernosus motoneurons, respectively. No significant difference was seen between mean somal cross sectional areas of motoneurons innervating bulbospongiosus and ischiocavernosus motoneurons or between external anal sphincter and external urethral sphincter motoneurons.
TABLE II Summary o f treatment effects on body weight and weight o f penis and attached muscles
Unweighted mean _+ S.E.M. aWeight of penis and attached muscles expressed as a percentage of body weight. Treatment
No. o f rats
Body weight (g)
Penis and muscle a
Sham C+TP C+P
12 13 17
402.1 + 10.3 392.7 + 7.8 380.2 ___6.9
0.43 + 0.01 0.45 + 0.03 0.20 + 0.01"
*P < 0.01; Sham vs. C+P comparison.
328 TABLE III Summary o f statistical analyses o f treatment effects on pudendal motoneuron somal cross sectional area
a See legend of Fig. 1 for explanation of abbreviations; b see text for formula; c degrees of freedom among subgroups from nested ANOVA see text; d established using Dunnet's test (two-tailed; ref. 5); n.s., not significant. Planned comparisons o f means
Motoneuron VBS DBS EAS IC EUS
Sham vs. C + T P
Sham vs. C + P
? (drY)
t b (df;e d)
1.195 1.831 1.119 0.623 0.450
2.893 2.723 0.164 3.809 0.762
(12; n.s.) ( 9; n.s.) (15; n.s.) (18; n.s.) (12; n.s.)
(12; ( 9; (15; (18; (12;
0.05) 0.05) n.s.) 0.01) n.s.)
Total body weight and the weight of the penis and attached muscles were analyzed to assess the effectiveness of the testosterone treatment. No significant difference in total b o d y weight was detected across t r e a t m e n t subgroups (Table II). Penis and attached muscle weight (expressed as percent of total body weight) was 50% smaller in the C + P subgroup c o m p a r e d to the sham subgroup ( P < 0.01; two tailed t-test), whereas, the sham and C + T P subgroups were statistically the same (Table II). Thus, the castration effectively r e m o v e d endogenous testosterone, and the castration induced atrophy of the penis and attached muscles was p r e v e n t e d by the testosterone pellet implants.
Somal
Cross Sectional
800700
L ..-.--
~I
Area Sham C+TP C+P
o
I VBS DBS EAS
IC
EUS
Fig. 1. A bar chart of mean somal cross sectional areas of ventral bulbospongiosus (VBS), dorsal bulbospongiosus (DBS), external anal sphincter (EAS), ischiocavernosus (IC), and external urethral sphincter (EUS) motoneurons in each treatment group is presented. The means are unweighted means, and the standard error bars were calculated using a random effects model. Significant testosterone treatment effects were detected in the cases of VBS, DBS, and IC motoneurons (Sham vs. C+P comparisons; ~P < 0.01; 2p < 0.05). See text for details concerning statistical tests.
Based on previously published observations 2'3'7n3't6, was anticipated that significant differences in motoneuron somal cross sectional areas may be detected between the sham and C + P treatment groups but not between the sham and C + T P t r e a t m e n t groups. Therefore, two planned comparisons, sham vs. C + P and sham vs. C + T P , were carried out on means in each of the five populations of m o t o n e u r o n s using a Student's t-test with the following formula28:
17 it
where m i and ni are the unweighted mean and n u m b e r respectively of all the observations within a t r e a t m e n t group, and MSsubgroups is the mean square among subgroups. D u n n e t t ' s test 5 for comparing two treatments to one control was done, and the degrees of f r e e d o m associated with the MSsubgroup values (•(ai-1); where a i is the number of rats in each treatment group) were used to determine significance (see Table III). Because there was significant variability between animals within treatments, a nested A N O V A consisting of t r e a t m e n t groups, rats in each t r e a t m e n t group (subgroups), and somal cross sectional area measurements o b t a i n e d from each rat was done for each population of m o t o n e u r o n s to estimate MSsubgroups, between rat variability, and within rat variability. The between rat variability and within rat variability were used to obtain an unbiased estimate of the variance of an observation, and this estimate was in turn used to obtain the estimate of the standard error of each mean. As can be seen in Fig. 1 and Table III, no difference in somal cross sectional area between the C + T P and sham t r e a t m e n t groups was detected within any of the populations of motoneurons. Significant decreases (1520%) in mean somal cross sectional areas were observed in the C + P c o m p a r e d to the sham treatment groups in the cases of dorsal and ventral bulbospongiosus (P < 0.05) and ischiocavernosus ( P < 0.01) motoneurons. In contrast, no treatment effect on m e a n somal size was detected for external anal sphincter and external urethral sphincter motoneurons. Thus, testosterone has a differential effect on somal size among p u d e n d a l m o t o n e u r o n s in adult male rats. The sexually dimorphic bulbospongiosus and ischiocavernosus m o t o n e u r o n s were sensitive to androgen treatment, whereas the non-sexually dimorphic external anal sphincter and external urethral sphincter m o t o n e u r o n s were not. The observed androgen dependence of bulbospongiosus m o t o n e u r o n somal size was expected and agrees with previously r e p o r t e d studies TM ~5-17. In the case of ischiocavernosus m o t o n e u r o n somal size, a similar d e p e n d e n c e upon androgen in a d u l t h o o d
329 was suspected, since, like bulbospongiosus m o t o n e u r o n s , ischiocavernosus m o t o n e u r o n s are sexually dimorphic. The lack of androgen sensitivity in the case of external anal sphincter m o t o n e u r o n s has b e e n previously suggested based u p o n indirect evidence obtained from testosterone-treated neonatal female rats 14. However, this is the first direct study of possible androgen effects on rat external anal sphincter and external urethral sphincter
rons throughout the spinal cord and brainstem accumulate androgen 23, and external anal sphincter and external urethral sphincter m o t o n e u r o n s do not exhibit this androgen dependence. F u r t h e r m o r e , the bulbospongiosus muscles accumulate androgen 29 and exhibit marked atrophy following castration 6'31'32. R a n d and Breedlove have reported that unilateral local application of testosterone to bulbospongiosus muscles in castrated adult
m o t o n e u r o n s in adulthood. The results of this study have important consequences for future experiments. It is clear that the D M and D L
male rats produces differential effects on D M m o t o n e u ron dendritic length and arbor shape 2°'21. Also, Araki et
m o t o n e u r o n s are heterogeneous not only with respect to target musculature but also with respect to androgen
toneurons axotomized and allowed to innervate grafted soleus muscle is no longer testosterone d e p e n d e n t ,
sensitivity in adulthood. Therefore, it is essential to positively identify individual populations of D M and D L
whereas D M m o t o n e u r o n s axotomized and allowed to reinnervate perineal muscles retain androgen sensitivity.
m o t o n e u r o n s when undertaking steroid m a n i p u l a t i o n studies. F r o m a practical perspective, external anal
Thus, the observed effect of testosterone deprivation on bulbospongiosus, as well as ischiocavernosus, motoneu-
sphincter or external urethral sphincter m o t o n e u r o n s can be used as a control population of m o t o n e u r o n s to differentiate between general effects of androgens on mo-
ron somal size may be secondary to atrophy of the target musculature as opposed to a direct effect at the level of the m o t o n e u r o n s .
toneurons and effects specific to 'penile m o t o n e u r o n s ' . These results also address the question of the site of action of testosterone in the m a i n t e n a n c e of bulbospongiosus and ischiocavernosus m o t o n e u r o n somal size. It is
al.1 have demonstrated that m e a n somal size of D M mo-
spongiosus m o t o n e u r o n s in adult male rats because autoradiographic studies indicate that m o t o n e u r o n s in the D M nuclei accumulate androgens 2'4. However, m o t o n e u -
We would like to thank L.M. Mendell for the use of his facilities; D. Malysz for technical assistance; N.R. Mendell for help with the statistical methodology; and K.R. Peshori for reading a previous version of the manuscript. This work was supported by R01NS24206 and BNS-9111207 (W.EC.) and a summer fellowship from the Undergraduate Research and Creative Activities Program, SUNY at Stony Brook (S.W.K.). Some assistance was also provided by P01-NS14899 and R01-NSI6996 to L.M. Mendell.
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