The Homeodomain Transcription Factors, Six6 and Six3, in the Circadian Regulation of Reproduction

项目摘要 昼夜节律对整体健康至关重要。扰乱昼夜节律(例如，由于倒班工作、时差、 夜间暴露在阳光下)会导致许多疾病，包括不孕不育。不孕不育影响15.5%的妇女 美国和世界上8%-12%的夫妇。不孕不育的负担很大，而且还与 社交功能障碍、焦虑和生活质量下降。最近的研究将倒班工作与异常联系在一起 月经周期、流产率增加和生殖问题。这突显了 在正常繁殖中有强烈的昼夜节律。视交叉上核(SCN)是主钟， 协调昼夜节律。SCN产生神经肽精氨酸加压素(AVP)和 血管活性肠肽(VIP)孤束核内的AVP和VIP神经元投射到下丘脑Kispeptin和 分别是控制生殖的GnRH神经元。病变对SCN的产生或抑制或 AVP或VIP的作用会导致生育力下降，生殖功能下降，以及迟钝的促黄体生成素激增。 促黄体生成素高峰是排卵所必需的，由中央核控制。Six6和Six3是同源结构域转录 对SCN的发育、功能以及正常的昼夜节律和生殖行为至关重要的因素。梅隆一家 实验室已经表明，6只基因敲除小鼠表现出昼夜节律紊乱、生育力下降和缺乏 AVP和VIP在SCN中的表达。正常昼夜节律和SCN中Si6的必要性 成年后的生殖仍然是未知的。我们实验室的初步研究表明，Six3条件 SCN基因敲除会导致昼夜节律紊乱和不育症，而Six3杂合子男性 生殖行为异常。因此，这笔赠款的目的是确定SCN中Six 6用于 昼夜节律和生殖，并在机制水平上研究Six6和Six3如何调节SCN 功能。第一个目标是检查在神经元后SCN中有条件缺失的六个6的小鼠 分化以确定成人SCN中的Six6是否是正常昼夜节律所必需的。这些实验 将使用车轮运行活动的行为分析，三重转基因小鼠与PER2：：荧光素酶小鼠使用 生物发光实时监测SCN功能，原位杂交检测昼夜节律 AVP、VIP、PER2和BMal1在SCN中的表达。第二个目标是利用这些老鼠来确定 SCN中的Six6是正常生育、正常发情周期和昼夜节律发生所必需的。 调节的黄体生成素峰值。第三个目标将检验Six6和Six3是直接转录因子的假设 AVP、VIP、PER2和BMal1。这些实验将使用瞬时转基因和定点突变。 检测Six6和Six3在调节昼夜节律和SCN中的充分性和必要性的研究 神经肽基因。这项建议将通过检测新基因来阐明SCN的功能机制 昼夜节律和生殖生物学的候选人，Six6和Six3。这些目标将提供改进的 对昼夜节律和不孕症的理解。

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.