Project Summary Circadian rhythms are essential for overall health. Disruption of circadian rhythms (e.g., from shift work, jet lag, exposure to light at night) result in many disease states, including infertility. Infertility affects 15.5% of women in the USA and 8-12% of couples worldwide. The burden of infertility is significant, and is also associated with social dysfunction, anxiety, and decreased quality of life. Recent studies associate shift work with abnormal menstrual cycles, increased rates of miscarriage, and reproductive issues. This highlights the importance of robust circadian rhythms in normal reproduction. The suprachiasmatic nucleus (SCN) is the master clock and coordinates circadian rhythms. The SCN produces the neuropeptides arginine vasopressin (AVP) and vasoactive intestinal peptide (VIP). AVP and VIP neurons in the SCN project to the hypothalamic kisspeptin and GnRH neurons, respectively, that control reproduction. Lesions to the SCN or inhibition of the production or actions of AVP or VIP, result in decreased fecundity, decreased reproductive function, and a blunted LH surge. The LH surge is necessary for ovulation and is gated by the SCN. Six6 and Six3 are homeodomain transcription factors important in SCN development, function and normal circadian and reproductive behaviors. The Mellon lab has shown that Six6 knockout mice exhibit disruptions in circadian rhythms, decreased fertility, and lack of AVP and VIP expression in the SCN. The necessity of Six6 in the SCN for normal circadian rhythms and reproduction in adulthood remains unknown. Preliminary studies from our lab demonstrate that Six3 conditional knock out in the SCN results in disrupted circadian rhythms and subfertility and Six3 heterozygote males have abnormal reproductive behaviors. Thus, this grant aims to determine the necessity of Six6 in the SCN for circadian rhythms and reproduction, and to examine at a mechanistic level how Six6 and Six3 regulate SCN function. The first Aim will examine mice that have Six6 conditionally deleted in the SCN after neuronal differentiation to determine if Six6 in the adult SCN is necessary for normal circadian rhythms. These experiments will use behavioral analysis of wheel running activity, triple transgenic mice with Per2::Luciferase mice using bioluminescence real-time monitoring of SCN function, and in situ hybridization to examine the circadian expression of Avp, Vip, Per2, and Bmal1 mRNA in the SCN. The second Aim will use these mice to determine if Six6 in the SCN is necessary for normal fertility, normal estrous cycling, and for the occurrence of the circadian- regulated LH surge. The third Aim will test the hypothesis that Six6 and Six3 are direct transcription factors for Avp, Vip, Per2, and Bmal1. These experiments will use transient transfections and site-directed mutagenesis studies to test both the sufficiency and necessity of Six6 and Six3 in the regulation of circadian and SCN neuropeptide genes. This proposal will elucidate mechanisms of SCN function by examining novel gene candidates, Six6 and Six3, in both circadian and reproductive biology. These aims will provide improved understanding of circadian rhythms and infertility.