Normally occurring intersexuality and testosterone induced plasticity in the copulatory system of adult leopard geckos Melissa M. Holmes a, * , Oliver Putz b , David Crews b , Juli Wade a,c a Neuroscience Program, 108 Giltner Hall, Michigan State University, East Lansing, MI 48824-1101, USA b Section of Integrative Biology, University of Texas at Austin, Austin, TX 78712, USA c Departments of Psychology and Zoology, Michigan State University, East Lansing, MI 48824, USA Received 27 August 2004; revised 1 November 2004; accepted 30 November 2004 Abstract The copulatory neuromuscular system of lizards is highly sexually dimorphic. Adult males possess bilateral penises called hemipenes, which are independently controlled by two muscles, the retractor penis magnus (RPM) and transversus penis (TPN). These structures are not obvious in adult females. However, in adult female leopard geckos (Eublepharis macularius ), testosterone induces hemipene growth. We investigated whether these structures develop de novo in adulthood or are histologically present as rudimentary structures in the female leopard gecko. We also investigated the extent of sexual dimorphisms and plasticity in the associated neuromuscular components. To do this, we compared copulatory morphology (sizes of hemipenes, RPM and TPN muscle fibers, and associated motoneurons, as well as motoneuron and RPM fiber number) in adult females treated with testosterone, control females, and control males. All of the geckos possessed hemipenes, RPMs and TPNs, but these structures were indeed vestigial in control females. Testosterone induced striking increases in hemipene and copulatory muscle fiber size in females, but not to levels equivalent to control males. In parallel, males with increased levels of androgenic activity had larger hemipenes, suggesting naturally occurring steroid-induced plasticity. Copulatory motoneurons were not sexually dimorphic in size or number, and these measures did not respond to testosterone. The data demonstrate that the copulatory system of leopard geckos, in which gonadal sex is determined by egg incubation temperature, differs from that of many species (both reptilian and mammalian) with genotypic sex determination. Indeed, the system is remarkable in that adult females have normally occurring intersex characteristics and they exhibit substantial steroid-induced morphological plasticity in adulthood. D 2004 Elsevier Inc. All rights reserved. Keywords: Androgen; Intersex; Leopard gecko; Sex difference; Sexual dimorphism; Temperature dependent sex determination; Testosterone Introduction Comparative studies involving neuromuscular systems offer a powerful means of elucidating mechanisms regulat- ing both sexual differentiation of and adult plasticity in the nervous system. The copulatory neuromuscular system is particularly useful in this regard. In mammals this system shows a striking degree of both sexual differentiation and adult plasticity. A clitoris forms instead of a penis in developing females, and the motoneurons and muscles of the masculine copulatory neuromuscular system regress (Breedlove et al., 2002). Survival and further development of the copulatory neuroeffectors in males is dependent on androgens, and the sensitivity to androgens remains into adulthood (Breedlove et al., 2002). Seasonal increases in testosterone (T) result in increased motoneuron soma and muscle fiber size and, in parallel, stimulate copulatory behavior (Forger and Breedlove, 1987; Hegstrom et al., 2002). In contrast, in females androgens cannot resurrect the penis, muscles, or motoneurons in adulthood (e.g., Breed- love and Arnold, 1983; Tobin and Joubert, 1991). Similar to mammals, lizards possess a sexually dimor- phic copulatory neuromuscular system (Holmes and Wade, 2004a; Ruiz and Wade, 2002). Male lizards possess two 0018-506X/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.yhbeh.2004.11.020 * Corresponding author. Fax: +1 517 432 2744. E-mail address: holmes22@msu.edu (M.M. Holmes). Hormones and Behavior 47 (2005) 439 – 445 www.elsevier.com/locate/yhbeh