Project Summary The long-term goal of the proposed research is to determine the central mechanisms by which undernutrition and realimentation impact reproduction through regulation of gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) secretion, using male and female sheep as an animal model. Puberty onset integrates various internal and external cues resulting in an increased release of GnRH from the hypothalamus that imparts the capacity for sexual maturation and reproductive success. Inadequate energy intake (undernutrition) has a significant negative impact on GnRH, and subsequently LH secretion, thereby delaying puberty onset. However, the central mechanisms responsible for the suppression of GnRH/LH secretion during undernutrition or the increase of GnRH/LH secretion following re-feeding (realimentation) remain largely unknown. Thus, the objectives of this proposal are 1) to determine the role that AgRP signaling plays in regulating GnRH and kisspeptin neurons during undernutrition and realimentation, and 2) to determine the role that microglia play in regulating GnRH and kisspeptin neurons during undernutrition and realimentation. In Aim 1, we will characterize changes in AgRP signaling in GnRH and kisspeptin neurons in feed-restricted (FR) sheep, examine the in vivo effect of AgRP immunoneutralization in the arcuate nucleus (ARC) of the hypothalamus on LH secretion in FR sheep, and characterize changes in AgRP signaling in GnRH and kisspeptin neurons in refed sheep. In Aim 2, we will characterize changes central immune signaling in GnRH and kisspeptin neurons in FR sheep, examine the in vivo effect of central infusion of an interleukin-1 receptor antagonist on LH secretion in FR sheep, characterize changes in central immune signaling in GnRH and kisspeptin neurons in refed sheep, and examine the in vitro effect of low glucose and insulin on microglia phenotype and function. Herein, with our expertise in whole-animal physiology, in vivo drug delivery, immunohistochemistry, and in vitro cell culture we have designed experiments to apply the highly innovative technique of a fluorescent in situ hybridization assay, RNAscope, for detection of signaling components for AgRP (Aim 1) and interleukin-1β (Aim 2). The proposed experiments will not only address the neuronal networks by which changes in metabolic state impact reproduction, but also provide important insight into the role the immune system likely plays. Thus, this work will provide a greater understanding of how undernutrition impacts central networks that regulate GnRH/LH secretion and yield novel and critical insight that may allow for better control of the timing of puberty, and ultimately lead to improved human health through prevention of disorders (e.g. cardiovascular disease and depression) associated with delayed puberty.