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.
