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

生殖昼夜节律调节中的同源域转录因子 Six6 和 Six3

• 批准号: 9905316
• 负责人: Lauren Elizabeth Chun
• 金额: $ 4.41万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-01 至 2020-11-05
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9905316
• 关键词: ARNTL gene; Adult; Affect; Anxiety; Argipressin; Behavior; Behavioral; Binding; Binding Sites; Biological Assay; Bioluminescence; Candidate Disease Gene; Cells; Circadian Dysregulation; Circadian Rhythms; Couples; Darkness; Data; Development; Disease; Electrophoretic Mobility Shift Assay; Estrous Cycle; Estrus; Exhibits; Exposure to; Fertility; Fibroblasts; GNRH1 gene; Genes; Genetic Transcription; Grant; Health; Heterozygote; Hypothalamic structure; In Situ Hybridization; In Vitro; Infertility; Jet Lag Syndrome; KISS1 gene; Knock-in; Knock-out; Knockout Mice; Lesion; Light; Luciferases; Menstrual cycle; Messenger RNA; Molecular; Molecular Abnormality; Mus; Neuronal Differentiation; Neurons; Neuropeptide Gene; Neuropeptides; Output; Ovulation; Periodicity; Plasma; Production; Promoter Regions; Proteins; Quality of life; Reproduction; Reproductive Behavior; Reproductive Biology; Reproductive Health; Reproductive History; Running; Site; Site-Directed Mutagenesis; Spontaneous abortion; Synapsins; System; Techniques; Testing; Time; Tissues; Transcriptional Regulation; Transfection; Transgenic Mice; Transgenic Organisms; United States; United States National Institutes of Health; Vasoactive Intestinal Peptide; Woman; circadian; circadian regulation; conditional knockout; experimental study; female fertility; homeodomain; improved; infertility treatment; knock-down; male; molecular clock; mouse model; novel; overexpression; promoter; protein expression; real time monitoring; reproductive axis; reproductive function; shift work; social deficits; subfertility; suprachiasmatic nucleus; temporal measurement; transcription factor; unpublished works

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.

项目概要 昼夜节律对于整体健康至关重要。昼夜节律紊乱（例如，轮班工作、时差反应、 夜间暴露在光线下）会导致许多疾病状态，包括不孕不育。不孕不育影响着15.5%的女性 美国和全球 8-12% 的夫妇。不孕不育的负担是巨大的，并且也与 社交功能障碍、焦虑和生活质量下降。最近的研究将轮班工作与异常联系起来 月经周期、流产率增加和生殖问题。这凸显了重要性 正常生殖过程中的稳健昼夜节律。视交叉上核 (SCN) 是主时钟， 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）。 SCN 中的 AVP 和 VIP 神经元投射到下丘脑 Kisspeptin 和 GnRH 神经元分别控制生殖。 SCN 损伤或产生或抑制 AVP 或 VIP 的作用会导致生育力下降、生殖功能下降和 LH 激增减弱。 LH 激增是排卵所必需的，由 SCN 控制。 Six6 和 Six3 是同源域转录 SCN 发育、功能以及正常昼夜节律和生殖行为的重要因素。梅隆 实验室表明，Six6 基因敲除小鼠表现出昼夜节律紊乱、生育能力下降和缺乏 AVP 和 VIP 在 SCN 中表达。 SCN 中 Six6 对于正常昼夜节律的必要性 成年后的繁殖情况仍然未知。我们实验室的初步研究表明 Six3 有条件 SCN 中的敲除会导致昼夜节律紊乱和生育力低下，而 Six3 杂合子男性 异常的生殖行为。因此，这笔赠款旨在确定 SCN 中 Six6 的必要性 昼夜节律和生殖，并在机制水平上检查 Six6 和 Six3 如何调节 SCN 功能。第一个目标将检查 SCN 中神经元被条件性删除后的小鼠。 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 将使用轮运行活动的行为分析，使用 Per2::Luciferase 小鼠的三重转基因小鼠 生物发光实时监测SCN功能，原位杂交检查昼夜节律 SCN 中 Avp、Vip、Per2 和 Bmal1 mRNA 的表达。第二个目标将使用这些小鼠来确定是否 SCN 中的 Six6 对于正常生育、正常动情周期以及昼夜节律的发生是必需的 调节 LH 激增。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染和定点诱变 检验 Six6 和 Six3 在昼夜节律和 SCN 调节中的充分性和必要性的研究 神经肽基因。该提案将通过检查新基因来阐明 SCN 功能的机制 昼夜节律和生殖生物学领域的候选人 Six6 和 Six3。这些目标将提供改进的 了解昼夜节律和不孕症。