Sex Differences in Fos Protein Expression in the Neonatal Rat Brain K. M. Olesen and A. P. Auger Department of Psychology, University of Wisconsin, Madison, WI, USA. Key words: sexual differentiation, oestrogen, steroid hormone, hypothalamus, development. Abstract Sex differences in the brain and behaviour are mostly a result of transient increases in testosterone during the perinatal period. Testosterone influences brain development primarily through aromatization to oes- tradiol and subsequent binding to oestrogen receptors. Although some studies report that steroid hor- mones regulate the expression of the inducible transcription factor, Fos, in developing brain, it is not known if there is a sex difference in Fos expression. Changes in Fos protein can be used as an indicator of neuronal/ genomic activity. Thus, it provides a useful tool to identify brain regions responding directly or indirectly to steroid hormones. In a first experiment, we examined Fos protein expression in the developing male and female rat brain using western immunoblotting. Dissections were taken from male and female rat pups on the day of birth (postnatal day 0; PN 0), PN1, PN5, PN11 or PN20. Although there was no difference on PN 0, males expressed significantly greater levels of Fos protein on PN1, PN5 and PN20. In a second experiment, we localized the sex difference in Fos protein expression using immunocytochemistry. We found that males expressed significantly higher levels of Fos within a variety of brain regions. These data indicate a sex difference in Fos protein expression during brain development, suggesting a potential role for Fos in differentiating male from female rat brain. Sex differences in brain and behaviour are largely a result of early steroid hormone action during brain development (1, 2), although recent studies suggest that steroid hormone-inde- pendent mechanisms play some role in sexually differentiating the brain (3). During brain development, male rats are exposed to high levels of testosterone, leading to masculini- zation and defeminization of the brain. On the other hand, the female brain is feminized by exposure to significantly lower levels of testosterone (4) and oestradiol (5). Altering the levels of steroid hormones during a critical period can sex reverse brain development. For example, castration of new- born male rats interferes with behavioural masculinization (increased male sexual behaviour) and behavioural defemini- zation (decreased female sexual behaviour) (6, 7). Similarly, administration of testosterone to newborn female rats results in behavioural masculinization and defeminization (8). These behavioural changes are likely due to the effect of testosterone on neuronal survival, neuronal migration and the plasticity of both neurones and glia (1, 2, 9). Steroid hormones produce transient and lasting changes within the brain mainly through their actions on intracellular steroid receptors. Upon steroid hormone binding, steroid receptors undergo a conformational change and form dimer complexes with other ligand bound receptors. Steroid recep- tors then bind to DNA and recruit additional proteins, including coactivators, to form a transcriptional complex. This complex then initiates gene transcription and thereby protein expression (10). Not only are steroid receptors themselves important in regulating brain differentiation, but additional factors, such as coactivators, recruited to the transcriptional complex are equally important (11, 12). Although much is known about the physiological and behavioural outcomes of steroid action, little is known about what signalling factors are altered by steroid hormones during brain differentiation. One transcription factor known to be regulated by steroid hormones is c-fos, which codes for Fos protein. Testosterone, oestradiol and progesterone, but not 5a-dihydrotestosterone, increase Fos protein expression in the developing and adult brain (13–16). Fos protein expression can also be up- regulated by neurotransmitters, such as dopamine (17), nonsteroid hormones, such as oxytocin (18), and a variety Correspondence to: Dr Anthony P. Auger, Psychology Department, University of Wisconsin, Madison, 1202 West Johnson Street, Madison, WI 53706, USA (e-mail: apauger@wisc.edu). Journal of Neuroendocrinology, 2005, Vol. 17, 255–261 doi:10.1111/j.1365-2826.2005.01302.x Ó 2005 Blackwell Publishing Ltd