Testosterone Regulates Androgen Receptor Immunoreactivity in the Copulatory, but not Courtship, Neuromuscular System in Adult Male Green Anoles M. M. Holmes* and J. Wade*† *Neuroscience Program and †Departments of Psychology and Zoology, Michigan State University, East Lansing, MI, USA. Key words: steroid hormone, motoneurone, muscle, season, lizard, reproduction, sexual dimorphism, sex difference. Abstract Androgens regulate the expression of male reproductive behaviour in diverse vertebrate species, often acting on androgen receptors (AR) to induce structural or functional changes in the nervous system and periphery. Male green anoles possess two sexually dimorphic neuromuscular systems, one controlling throat fan (dewlap) extension, which occurs during courtship, and the other mediating copulatory organ function. Although androgens are required for behavioural activation in both systems, testosterone has differential effects on the neuromuscular morphology. It increases the size of copulatory muscle fibres during the breeding season, but significant effects on dewlap muscle fibre size and motoneurone soma size in either system have not been detected. Corresponding to the lack of testosterone-induced morphological effects in the courtship system, relatively low levels of AR are expressed in the associated motoneurones. The present experiment had two goals, aiming to determine whether: (i) the other courtship and copulatory neuro- muscular tissues express AR and (ii) testosterone and/or seasonal environmental changes regulate AR expression. The percentage of AR+ nuclei was evaluated in both the breeding and nonbreeding seasons in gonadally intact adult males (Experiment 1) and in castrated males treated with either testosterone or vehicle (Experiment 2). AR was extensively expressed in the dewlap and copulatory muscles, and in a high percentage of the copulatory motoneurones, but immunoreactivity did not vary across season. Testosterone increased the percentage of AR+ nuclei in the copulatory muscles of both breeding and nonbreeding males but not in the dewlap muscle or copulatory motoneurones. Finally, the target structures for both systems (cartilages and hemipenes) expressed AR in all animals. Therefore, the effects of testosterone on AR immunoreactivity suggest that up-regulation of the receptors may be important for morphological change. However, because all structures investigated in the present experiment expressed AR, the data also indicate that the receptors are involved with other functions. Androgens commonly regulate the expression of masculine reproductive behaviours in a wide variety of vertebrate groups, including fish (1), frogs (2), birds (3, 4) and mammals (5). Although the motivation for such behaviours is mediated by hypothalamic and preoptic brain regions (6), the execution of courtship and copulatory behaviours requires neuromus- cular systems, which are frequently sexually dimorphic (7). Androgens serve not only to activate the behaviours in adulthood, but often also mediate the development and maintenance of the underlying neuromuscular components (7). These effects on motoneurone and muscle morphology in particular usually occur by direct effects of androgenic steroids on the androgen receptor (AR) (7). As such, determining the distribution and regulation of AR is critical for understanding mechanisms that regulate structural and functional change in reproductive neural circuits. Green anoles (Anolis carolinensis) are a particularly useful model species with which to study steroid hormone effects on structure and function because they possess two sexually dimorphic neuromuscular systems. One is involved in court- ship behaviour in this lizard, which includes extension of a red throat fan called a dewlap. Although both sexes possess dewlaps, they are approximately seven-fold larger in males than females (8), and males extend their dewlaps far more frequently than females (9–11). Paralleling the sex difference in size and overall use of the dewlap, the neuromuscular components that mediate its extension are larger in males than females. Specifically, extension of the dewlap is caused Correspondence to: Dr Melissa M. Holmes, Neuroscience Program, 108 Giltner Hall, Michigan State University, East Lansing, MI, 48824-1101, USA (e-mail: holmes22@msu.edu). Journal of Neuroendocrinology, 2005, Vol. 17, 560–569 doi:10.1111/j.1365-2826.2005.01339.x Ó 2005 Blackwell Publishing Ltd