ORIGINAL ARTICLE Kisspeptin Cells in the Ovine Arcuate Nucleus Express Prolactin Receptor but not Melatonin Receptor Q. Li, A. Rao, A. Pereira, I. J. Clarke and J. T. Smith Department of Physiology, Monash University, Clayton, Victoria, Australia. The seasonality of reproduction ensures the birth of young at the optimal time of year. Similar to most animals, sheep are seasonal breeders, experiencing annual periods of reproductive quiescence and renaissance that are controlled by environmental cues. Animals use photoperiod to perceive the change of seasons and the trans- duction of photoperiodic information to reproductive neuroendo- crine activity is mediated by melatonin, which is secreted by the pineal gland at night. In both ewes and Syrian hamsters, removal of the pineal gland prevents the response to photoperiod, in terms of reproduction, and this can be restored with melatonin adminis- tration (1–5). Although photoperiodic regulation of reproduction is a well-known characteristic of most animal species, the molecular and cellular mechanisms by which melatonin causes this seasonal change is not clear. Reproduction is driven by the pulsatile secretion of gonadotro- phin-releasing hormone (GnRH), which provides stimulus to pitui- tary gonadotropes. During the nonbreeding (anoestrous) season, luteinising hormone (LH) pulse frequency is reduced (6–8). Thus, GnRH ⁄ LH pulse frequency appears to be the over-riding determi- nant of seasonal breeding. During the nonbreeding season, the reduced LH pulse frequency is a result of enhanced sensitivity to the negative-feedback effects of oestrogen (6, 7). This is not a Journal of Neuroendocrinology Correspondence to: J. T. Smith, Department of Physiology, Building 13F, Monash University, Clayton, Victoria 3880, Australia (e-mail: jeremy.smith@monash.edu). Melatonin is secreted at night by the pineal gland and governs the reproductive system in sea- sonal breeders, such as sheep. The mechanism by which melatonin regulates reproduction is not known. The circannual rhythmicity of other factors, including prolactin, is also regulated by pho- toperiod via changes in melatonin secretion. In sheep, plasma prolactin levels are higher in the nonbreeding season than the breeding season. Kisspeptin, synthesised by neurones in the ovine arcuate nucleus (ARC) and preoptic area, is a key regulator of reproduction through stimulation of gonadotrophin-releasing hormone secretion and its expression in the ARC is reduced during the nonbreeding season. We hypothesised that kisspeptin expression is directly, or indirectly, regulated by melatonin and ⁄ or prolactin. We first examined the expression of melatonin receptor (MTNR1A) in kisspeptin (Kiss1 mRNA) neurones in the ARC of ovariectomised (OVX) sheep using double-label in situ hybridisation. MTNR1A mRNA was not expressed by kisspeptin neurones, whereas strong expression was detected in the pars tuberalis. We then examined the expression of the long-form prolactin receptor (PRLR-L) in ARC kisspeptin neurones. In OVX ewes, approxi- mately 60% of kisspeptin neurones expressed PRLR-L mRNA at similar levels in the breeding and nonbreeding seasons. We then aimed to determine whether prolactin treatment during the breeding season regulates kisspeptin expression in the ARC. Continuous central infusion of pro- lactin (20 lg ⁄ h for 7 days) in oestradiol-treated OVX sheep did not alter Kiss1 mRNA expression or luteinising hormone secretion, although it induced substantial phosphorylated signal trans- ducer and activator of transcription 5-immunoreactive nuclei staining in the mediobasal hypo- thalamus. We conclude that the seasonal change in kisspeptin neurones cannot be regulated directly by melatonin, although it may be a result of changes in prolactin levels. Despite this, kisspeptin expression was unchanged after exogenous prolactin treatment in breeding season ewes. Key words: kisspeptin, Kiss1, melatonin, prolactin, season. doi: 10.1111/j.1365-2826.2011.02195.x Journal of Neuroendocrinology 23, 871–882 ª 2011 The Authors. Journal of Neuroendocrinology ª 2011 Blackwell Publishing Ltd Journal of Neuroendocrinology From Molecular to Translational Neurobiology