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Proc. Nati. Acad. Sci. USA Vol. 83, pp. 7527-7531, October 1986 Neurobiology Sexual dimorphism in and canine spinal cord: Role of early androgen NANCY G. FORGER AND S. MARC BREEDLOVE Department of Psychology and Group in Endocrinology, University of California, Berkeley, CA 94720 Communicated by Frank A. Beach, June 16, 1986

ABSTRACT Onuf's nucleus, located in the sacral spinal as the external anal and urethral sphincters. The BC and IC cord of dogs, , and primates, innervates perineal muscles muscles attach to the penis in males and play important roles involved in copulatory behavior. A sexual dimorphism in in and in , and penile in Onuf's nucleus was found in humans and dogs: males have dogs and rats (18-21). In female dogs and humans, the IC significantly more motoneurons in this nucleus than do females. muscle is considerably smaller than in males and the BC Prenatal androgen treatment of female dogs eliminated the forms circular muscles serving to constrict the (22, dimorphism. In the homologous nucleus in rats, a similar effect 23). Contractions of the BC homologue have been recorded of androgen has been shown to involve sparing of motoneurons in female dogs during copulation and genital stimulation (24). from cell death. These results establish a morphological sex Here we describe a sex difference in the number of motoneu- difference in a human central region of known rons comprising Onuf's nucleus ofhumans and dogs and report function; well-studied animal models suggest explanations of further that the canine dimorphism can be eliminated by treating the development of this dimorphism. females with androgen early in development. Sex differences in the behavior of are ubiquitous and have long been recognized, but only recently have sexual MATERIALS AND METHODS dimorphisms been identified in (CNS) areas mediating sexually dimorphic behaviors. These ana- Dogs. Spinal cords and perineums were obtained from adult tomical differences can be striking and include dimorphism in purebred beagles that had served as subjects in behavioral the type and number of synapses and large regional sex studies unrelated to the current report (25). Normal females differences in cell size or number (1, 2). In several cases (n = 4) and males (n = 4) were compared with five androgenized sexual dimorphisms in CNS morphology have been shown to females born to dams that received daily injections of develop under hormonal control. For example, the sex testosterone propionate (1.1 mg/kg of dam's body weight) difference in the size of nuclei involved in copulatory behav- during days 24-43 postcoitum. Four of these females also ior of rats and sexually dimorphic song production in zebra received a 37.5-mg subcutaneous implant of crystalline finches is diminished by treating females with androgen testosterone on the day of birth. The implant was placed during early development (3, 4). Neonatal androgen treat- dorsally beneath the skin of the and was gradually ment of female rats also attenuates the dimorphism in the resorbed during the first 30-40 postnatal days. sexually dimorphic nucleus of the preoptic area and in the The dogs were sacrificed as adults (3-6 yr) and perfused pattern of connectivity in the preoptic area (5, 6). with saline and formalin. Perineums were dissected and Until quite recently, the question of whether similar examined for muscular morphology. Spinal cords were em- differences exist in the CNS of humans has not been bedded in paraffin, sectioned at 15 ,um, and treated with a addressed. Swaab and Fliers (7) reported a sexual dimor- Kluver-Barrera stain. Bilateral counts in every third section phism in the volume and cell number of a nucleus in the were made of all motoneurons within the Onuf's nucleus preoptic area of humans, comparable to findings in rats. The function of the sexually dimorphic portion of the preoptic halo. The resulting sum for each dog was tripled to estimate area has not been established in either species (8), although the total number of Onuf's nucleus motoneurons. Counts surrounding neural areas in rats are involved in the regulation were corrected for split somas by the method of Konigsmark of male sexual behavior and cyclic release of gonadotropins (26). In addition, six camera lucida tracings were made at set (9). In the present study we have identified a sex difference intervals through each dog's cord of the halo defining Onuf's in humans and dogs in a spinal nucleus known to be involved nucleus; mean cross-sectional halo area and the total volume in copulatory behavior. of Onuf's nucleus were determined from these tracings. Onuf's nucleus, in the ventral horn ofthe sacral spinal cord, Somas of motoneurons in 10 representative halos from each is morphologically very similar in humans, monkeys, cats, and dog were also drawn to estimate mean size. All counts dogs (10-14). It consists of a slender column of small-to- and measurements were made by an experimenter "blind" to medium-sized motoneurons that stands out sharply in - group membership of the sections being examined. stained coronal sections as a pale area, or "halo," demarcated Humans. Spinal cords of eight human females and nine from surrounding myelinated fibers. The halo apparently results males from the Yakovlev collection of the Armed Forces from an intense concentration of longitudinally running Institute of Pathology were examined. Subjects died of from motoneurons within the nucleus (15). causes unrelated to spinal cord function; ages at Human neurological studies and retrograde labeling stud- ranged from 2 to 87 yr in females (mean + SEM, 22 ± 10 yr) ies in cats and dogs (11-14, 16, 17) show that Onufs nucleus and 1.5 mos to 60 yr in males (mean + SEM, 22 + 8 yr). Each innervates striated perineal muscles including the bulbo- cord was embedded in celloidin and cut coronally at 35 ,um; cavernosus (BC) and ischiocavernosus (IC) muscles as well and every 5th (n = 1), 10th (n = 10), or 20th (n = 6) section

The publication costs of this article were defrayed in part by page charge Abbreviations: CNS, central nervous system; BC, bulbocavernosus; payment. This article must therefore be hereby marked "advertisement" IC, ischiocavernosus; VL, ventrolateral; DM, dorsomedial; SNB, in accordance with 18 U.S.C. §1734 solely to indicate this fact. spinal nucleus of the BC muscle.

7527 Downloaded by guest on September 26, 2021 7528 Neurobiology: Forger and Breedlove Proc. Natl. Acad. Sci. USA 83 (1986) A

FIG. 1. Coronal Kliver-Barrera-stained section through Onuf's nucleus in the sacral spinal cord of a female dog exposed to androgen early in development. (A) The arrow points to Onuf's nucleus, which stands out as a pale, oval area devoid of myelinated fibers. (B) Higher-power magnification of the nucleus in A.

was mounted and Nissl-stained. The sampling ratio was well RESULTS matched between sexes. Adjacent Weigert-stained sections aided nucleus identification. Dogs. Fig. 1 depicts canine Onufs nucleus in which both Motoneurons comprising Onufs nucleus were counted in halo and individual motoneurons are clearly visible. Male Nissl-stained sections, and the sum was adjusted according to dogs had significantly more in Onuf s nucleus than the sampling ratio to estimate total motoneuronal number. did females (P < 0.02, two-tailed t test) (Fig. 2A). Onuf s

A B 4500

3900 1.2 E C 3300 0 1.0 2700 0.8 _

0 2100F i 0.62 nH ~~~~~0.4 0.2

d Y Y?+TP 6 ? ?+TP FIG. 2. The number of motoneurons in Onuf's nucleus (A) and the bilateral volume of Onuf's nucleus (B) in male, female, and androgen-treated female dogs. Bars represent means + SEM. Dashed lines in A indicate uncorrected counts, while solid lines are counts corrected for split somas. Females had significantly fewer motoneurons and smaller nuclear volumes than did males. Females treated with androgen early in development did not differ significantly from males in motoneuron number and had more neurons and larger nuclear volumes than did untreated females. TP, testosterone propionate. Downloaded by guest on September 26, 2021 Neurobiology: Forger and Breedlove Proc. Natl. Acad. Sci. USA 83 (1986) 7529 nucleus of androgenized females was masculinized; moto- position as well as somewhat larger cell size (Fig. 3). Human number was not different from that of males (P > males had significantly more motoneurons in the VL portion 0.25) and was significantly greater than the number of ofOnufs nucleus than did females (P < 0.025; Fig. 4). No sex motoneurons in normal females (P < 0.05). These differences difference was observed in the few DM cells seen. Motoneu- were observed for raw motoneuronal counts and for counts ron number was not correlated with the age of the subject in corrected for split somas. Perineal musculature of androgen- either cell population for either sex. No sex difference in treated females also was markedly masculinized: external estimated nucleus length was observed. genitalia consisted of a penis with BC and IC muscles surrounding the base of the phallus, as in males. Masculin- DISCUSSION ization of the perineum and spinal cord of the androgenized female receiving testosterone propionate prenatally only was A sex difference was found in the number of motoneurons in as complete as for the four females receiving testosterone at Onuf s nucleus of dogs and in VL Onufs nucleus ofhumans; birth in addition to the prenatal treatment. Sexual differen- early androgen treatment eliminated the dimorphism in dogs. tiation of the CNS probably occurs prenatally in dogs; As in humans, Onuf's nucleus of cats is divided into two cell postnatal testosterone treatment was initiated for unrelated groups. Retrogradely transported fluorescent dye labels cells behavioral studies (25) and apparently did not influence in VL Onufs nucleus after injection of the sexually dimor- spinal organization. phic IC muscle of cats, while DM cells are labeled after The total bilateral volume of Onuf's nucleus (mean cross- injection of the external anal sphincter (28). Therefore, the sectional area x the length of the nucleus) was 2.2 times human sex difference in -motoneuron number was found in greater in male dogs than in female dogs (P < 0.01) (Fig. 2B). that portion of Onuf s nucleus that, in cats, innervates The volume of Onufs nucleus in androgenized females was sexually dimorphic muscles. While this study was in intermediate and differed significantly from both males and progress, a comparable sex difference in the number of untreated females (P < 0.05). Mean motoneuronal soma size Onuf s nucleus motoneurons labeled by applying horseradish was not significantly different in the three groups. peroxidase to the pudendal of macaques was reported Humans. Onufs nucleus in humans is divided into two cell (29). groups (27). The ventrolateral (VL) group was consistently The spinal nucleus of the BC muscle (SNB) of rats is present and was composed of many small, densely staining homologous to Onufs nucleus in that it innervates the BC. cells. A dorsomedial (DM) group was only intermittently Male rats have many more SNB motoneurons than do identifiable and could be distinguished on the basis of females (30). Although females are born with the perineal B

FIG. 3. Nissl-stained sections through Onufs nucleus in humans. The arrow in A points to VL Onuf s nucleus; no DM cells were observed in this section. Both VL and DM cell groups can be identified in B. Downloaded by guest on September 26, 2021 7530 Neurobiology: Forger and Breedlove Proc. Natl. Acad Sci. USA 83 (1986)

A B

VL DM 2400 - T _ 2000 4000 E 0 u 2 1600 I1 3000 0 0 U 1200 2000 800 _-

1000 4001-

I I 9 1y d I FIG. 4. (A) The number of motoneurons in the VL and DM cell groups of Onuf's nucleus in humans. Males had more motoneurons in the VL nucleus than did females (P < 0.025). There was no significant sex difference in the number of DM cells (A) or in the rostrocaudal extent of Onufs nucleus (nucleus length in tum) (B). muscles and motoneurons innervating them, both the mus- 1. Arnold, A. P. & Gorski, R. A. (1984) Annu. Rev. Neurosci. 7, cles and motoneurons die shortly after birth. BC muscles and 413-442. SNB cells can be spared in female rats, however, by perinatal 2. DeVries, G. J., DeBruin, J. C. P., Uylings, H. B. M. & Cor- treatment with androgen (3). Evidence suggests that andro- ner, M. A., eds. (1984) Progress in Research (Elsevier, muscles to Amsterdam), Vol. 61, Sect. 2, pp. 129-184. gen acts on the perineal enhance motoneuron 3. Breedlove, S. M. & Arnold, A. P. (1983) J. Neurosci. 3, survival (31, 32). 424-432. The sexual dimorphism in the number of perineal 4. Gurney, M. E. & Konishi, M. (1980) Science 208, 1380-1382. motoneurons is less pronounced in dogs and humans than it 5. Gorski, R. A., Gordon, J. H., Shryne, J. E. & Southam, is in rats (30), as might be expected because female humans A. M. (1978) Brain Res. 148, 333-346. and dogs retain perineal muscles (modified in form compared 6. Raisman, G. & Field, P. M. (1973) Brain Res. 54, 1-29. with those of males), while the muscles involute in female 7. Swaab, D. F. & Fliers, E. (1985) Science 228, 1112-1115. rats. Although we cannot be certain how this sex difference 8. Arendash, G. W. & Gorski, R. A. (1983) Brain Res. Bull. 10, in the human spinal cord comes about, the studies of Onuf's 147-154. nucleus in and the SNB in rats a critical role of 9. Hart, B. L. & Leedy, M. G. (1985) in Handbook ofBehavioral dogs suggest Neurobiology, eds. Adler, N., Pfaff, D. & Goy, R. W. (Plen- perinatal androgen, as early administration of androgen um, New York), Vol. 7, pp. 373-422. reduces or eliminates the sex difference in motoneuronal 10. Onuf, B. (1899) J. Nerv. Ment. Dis. 26, 498-504. number. Furthermore, androgen causes the sex difference in 11. Nakagawa, S. (1980) Brain Res. 191, 337-344. rats by rescuing SNB cells that otherwise would die during 12. Thuroff, J. W., Bazeed, M. A., Schmidt, R. A., Luu, D. H. & the normal period of motoneuronal death (33, 34). We Tanagho, E. A. (1982) Urol. Int. 37, 110-120. recently have identified a decline in the total number ofspinal 13. Sato, M., Mizuno, N. & Konishi, A. (1978) Brain Res. 140, 149-154. motoneurons in human fetuses ofboth sexes (35) that occurs 14. Yamamoto, Y., Sato, M., Mizuno, N., Itoh, K., Nomura, S. & during the time ofandrogen production by males (36, 37). The Sugimoto, T. (1978) Neurosci. Lett. 7, 41-47. testicular secretion of androgen by human male fetuses may 15. Konishi, A., Satomi, H., Ise, H., Takatama, H. & Takahashi, spare some motoneurons in Onuf's nucleus from cell death, K. (1978) Brain Res. 156, 333-338. resulting in the greater number of neurons in adult males. 16. Onuf, B. (1900) Arch. Neurol. Psychopathol. 3, 387-412. Interestingly, Onuf's nucleus is particularly resistant to the 17. Mannen, T., Iwata, M., Toyokura, Y. & Nagashima, K. (1977) motoneuronal degeneration characteristic of amyotrophic J. Neurol. Neurosurg. Psychiatry 40, 464-469. 18. Karacan, I., Aslan, C. & Hirshkowitz, M. (1983) Science 220, lateral sclerosis (17). If this resistance is a residuum of 1080-1082. steroidal inhibition ofcell death during development, then the 19. Sachs, B. D. (1982) J. Reprod. Fertil. 66, 433-443. degeneration of these perineal motoneurons in females may 20. Hart, B. L. & Melese-d'Hospital, P. Y. (1983) Physiol. Behav. provide a model for the pathology of amyotrophic lateral 31, 807-813. sclerosis. 21. Hart, B. L. (1972) Anat. Rec. 173, 1-6. 22. Francis, C. C. (1952) The Human (Mosby, St. Louis, We thank Darrell Hall and Karen Rasmussen for technical assis- MO), pp. 98-105. tance. We gratefully acknowledge the contribution by Frank Beach 23. Miller, M. E., Christensen, G. & Evans, H. (1964) of and Mike Buehler of the beagles used in this study and the Armed the Dog (Saunders, Philadelphia), pp. 763-794. Forces Institute of Pathology for use of the Yakovlev collection and 24. Hart, B. L. (1970) Horm. Behav. 1, 93-104. photomicrographs of human material. This work was supported by 25. Ranson, E. & Beach, F. A. (1985) Horm. Behav. 19, 36-51. National Institutes of Health Grant NS19790 and National Science 26. Konigsmark, B. W. (1970) in Contemporary Research Meth- Foundation Grant BNS8451367. ods in , eds. Nauta, W. J. H. & Ebbeson, Downloaded by guest on September 26, 2021 Neurobiology: Forger and Breedlove Proc. Nati. Acad. Sci. USA 83 (1986) 7531

S. 0. E. (Springer, New York), pp. 315-340. 32. Breedlove, S. M. (1985) Soc. Neurosci. Abstr. 11, 160. 27. Schroder, H. D. (1981) Anat. Embryol. 162, 443-453. 33. Breedlove, S. M. (1984) Soc. Neurosci. Abstr. 10, 927. 28. Kuzuhara, S., Kanazawa, I., Nakanishi, T. (1980) Neurosci. 34. Nordeen, E. J., Nordeen, K. W., Sengelaub, D. R. & Arnold, Lett. 16, 125-130. A. P. (1985) Science 229, 671-673. 29. Ueyama, T., Mizuno, N., Takahashi, O., Nomura, S., 35. Forger, N. G. & Breedlove, S. M. (1986) Soc. Neurosci. Arakawa, H. & Matshushima, R. (1985) J. Comp. Neurol. 232, Abstr. 12, 984. 548-566. 36. Siiteri, P. K. & Wilson, J. D. (1974) J. Clin. Endocrinol. 30. Breedlove, S. M. & Arnold, A. P. (1980) Science 210, 564-566. Metab. 38, 113-124. 31. Fishman, R. B. & Breedlove, S. M. (1985) Soc. Neurosci. 37. Kaplan, S. L. & Grumbach, M. M. (1978) Clin. Endocrinol. Abstr. 11, 530. Metab. 7, 487-511. Downloaded by guest on September 26, 2021