PROJECT SUMMARY Puberty is a remarkable time during which the body develops secondary sexual characteristics and becomes capable of reproduction. Puberty onset is due to a complex interplay of factors, including genetic influences. Genetic mutations in disorders of puberty, including central precocious puberty (CPP) and delayed puberty, provide windows into understanding the neuroendocrine mechanisms of puberty and reproduction. The most common genetic cause of precocious puberty is due to loss-of-function mutations in Makorin Ring Finger Protein 3 (MKRN3), and accounts for ~40% of familial CPP. The function and regulation of MKRN3 are not well understood, but its protein structure suggests E3 ubiquitin ligase and RNA binding activities. Expression of Mkrn3 is high in the mouse hypothalamus and rapidly declines before puberty onset. This supports its role as the first identified inhibitor of puberty onset, hypothesized to act upstream of GnRH and/or its activators, such as kisspeptin. The current proposal aims to explore if alterations in Mkrn3 expression can similarly lead to delayed pubertal onset using innovative mouse models and human investigation. Preliminary studies demonstrate a delayed puberty phenotype in wild type female mice injected intracerebroventricularly with a recombinant virus overexpressing Mkrn3. This current model will be used to explore MKRN3’s mechanism of action, including its impact on known neuroendocrine players in reproduction. This recombinant virus will be used to assess if Mkrn3 overexpression can also lead to hypogonadotropic hypogonadism postpubertally following bilateral stereotaxic injection into the arcuate nuclei of the hypothalamus. In a more specific model of overexpression, Mkrn3 will be selectively overexpressed in kisspeptin neurons using a novel transgenic mouse model to better identify MKRN3’s targets. Additionally, given the observation in preliminary studies that increases in Mkrn3 expression can delay puberty onset, a well-characterized cohort of children with delayed puberty will be screened for coding and non-coding variants in MKRN3 as this has not been previously explored. Delayed puberty puts children at risk for long-term health risks. Therefore, elucidating the impact and mechanisms of action of MKRN3 in delaying pubertal onset has important implications for advancing child health, including improving diagnosis and management of pubertal disorders and can serve as a future therapeutic target. Using the proposed tools to identify MKRN3’s targets of action is critical to understanding this key player in the neuroendocrine control of puberty and reproduction.