SEX DIFFERENCES IN THE BRAIN OF GOLDFISH: GONADOTROPIN-RELEASING HORMONE AND VASOTOCINERGIC NEURONS I. S. PARHAR, a * H. TOSAKI, b Y. SAKUMA a and M. KOBAYASHI b a Department of Physiology, Nippon Medical School, Sendagi 1-1-5, Bunkyo-ku, Tokyo 113-8602, Japan b Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, University of Tokyo, Bunkyo, Tokyo 113-8657, Japan AbstractöThe di¡erences between male and female behaviors are re£ected in sexual dimorphism of brain structures and are found throughout the nervous system in a variety of vertebrates. The present study examined neurons immunolabeled for gonadotropin-releasing hormone and arginine vasotocin in the brain of the gold¢sh Carassius auratus to determine if these neurons are sexually dimorphic. There was no sex di¡erence or in£uence of sex steroids on the neuronal volume and optical density of staining of arginine vasotocin neurons. Similarly, gonadotropin-releasing hormone neurons of the terminal nerve and midbrain tegmentum did not di¡er between sexually mature males, females and maturing females replaced with sex steroids with respect to distribution, numbers, optical density of staining, or gross morphology. In maturing females, testosterone speci¢cally recruited additional preoptic gonadotropin-releasing hormone neurons to equal those in sexually mature individuals. Since estrogen had no e¡ect, the in£uence of testosterone on gonado- tropin-releasing hormone neuronal numbers appears to be independent of aromatization. Speci¢cally, the preoptic gonadotropin-releasing hormone neuronal size was signi¢cantly larger in sexually mature males than females. 11-Keto- testosterone-replacement to ovariectomized maturing females induced male-typical secondary characters and male-type courtship behavior but did not masculinize the preoptic gonadotropin-releasing hormone neuronal size. Our results show that the sexually dimorphic preoptic gonadotropin-releasing hormone neuronal size is determined by factors (genetic) other than gonadal steroids. Further, we propose the hypothesis that phenotypic and behavioral sex di¡erences need not be accompanied by structural di¡erences in gonadotropin-releasing hormone and arginine vasotocin in the brain. ß 2001 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: teleosts, sex steroids, ovariectomy, midbrain, preoptic area, terminal nerve. Sex di¡erences in the neural control of reproductive functions and sexual behaviors are a consequence of hor- mones secreted by the gonads. The in£uence of sex ste- roid hormones on the neural tissue is classically subdivided into organizational and activational e¡ects. It is generally believed that sex steroids exert an organ- izational in£uence on the developing brain during a restricted period of neural di¡erentiation. However, there is a growing body of evidence showing e¡ects of sex steroids on reversible neuroanatomical plasticity in the adult brain (McEwen, 1999). Sex di¡erences in the adult brain are often categorized in terms of volume of brain nuclei, cell numbers, cell density or size of individ- ual neurons. Not all sexual dimorphisms in neuronal numbers favor the male sex, and not all anatomical sex di¡erences are restricted to the hypothalamus (Matsumoto et al., 2000). However, as a rule, the sexu- ally dimorphic structures including the sexually dimor- phic nucleus of the preoptic area (POA) in mammals and the song-control nuclei in birds is larger and con- tains more cells in males than in females (Pilgrim and Hutchison, 1994; Rhodes and Rubin, 1999). In most vertebrates, including teleosts, gonadotropin- releasing hormone (GnRH) and arginine vasotocin (AVT) have been implicated in the control of reproduc- tion and reproductive behaviors (Foran and Bass, 1999). The development of preoptic GnRH neurons is tightly coupled to the ¢rst appearance of gonadotropes and GnRH projections in the pituitary of bony¢sh (Parhar, 1997), thus, indicating their relationship to reproduction. In teleosts, GnRH and AVT-producing neurons directly innervate the pituitary gland (Batten et al., 1990) and initiate a cascade of events leading from gonadotropin secretion to the onset of sexual maturation. Further- more, these two neuropeptides have been implicated in sexual plasticity in bony¢sh (Foran and Bass, 1999). GnRH neuronal plasticity is most obvious in several sex-changing ¢sh species. For example, in the protogy- 1099 *Corresponding author. Tel. : +81-3-3822-2131 ; fax : +81-3-5685- 3055. E-mail address : ishwar@nms.ac.jp (I. S. Parhar). Abbreviations : ABC, avidin^biotin complex; ANOVA, analysis of variance ; AVP, arginine vasopressin ; AVT, arginine vasotocin ; B, blank ; E, estradiol ; GnRH, gonadotropin-releasing hormone ; GSI, gonadosomatic index ; K, 11-ketotestosterone ; MB, mid- brain tegmentum ; MS222, tricaine methanesulfonate ; OVX, ovariectomized ; PBS, phosphate-bu¡ered saline ; POA, preoptic area ; SHAM or S, sham-operated ; T, testosterone ; TN, terminal nerve. NSC 4998 5-7-01 www.elsevier.com/locate/neuroscience Neuroscience Vol. 104, No. 4, pp. 1099^1110, 2001 ß 2001 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved PII:S0306-4522(01)00153-1 0306-4522 / 01 $20.00+0.00