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shift work; social deficits; subfertility; suprachiasmatic nucleus; temporal measurement; transcription factor; unpublished works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.

项目摘要 昼夜节律对整体健康至关重要。昼夜节律的破坏（例如，轮班工作，时差 夜间暴露于光）导致许多疾病状态，包括不孕症。15.5%的妇女患有不孕症， 美国和全世界8-12%的夫妇。不孕症的负担是显著的，并且还与以下因素有关： 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 月经周期，流产率增加和生殖问题。这突出了以下方面的重要性： 在正常的生殖过程中有很强的昼夜节律。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）和精氨酸加压素（AVP）。 血管活性肠肽（VIP）。SCN内AVP和VIP神经元投射到下丘脑kisspeptin， GnRH神经元，分别控制生殖。SCN病变或抑制生产或 AVP或VIP的作用导致生殖力下降、生殖功能下降和LH峰减弱。 LH峰是排卵所必需的，由SCN门控。Six 6和Six 3是同源结构域转录 SCN发育、功能和正常昼夜节律及生殖行为中的重要因素。梅隆 实验室已经表明，Six 6基因敲除小鼠表现出昼夜节律紊乱，生育能力下降，缺乏 SCN中AVP和VIP的表达。SCN中Six 6对正常昼夜节律的必要性， 成年后的生殖仍然是未知的。我们实验室的初步研究表明，Six 3条件 SCN中的敲除导致昼夜节律紊乱和生育力低下，并且Six 3杂合子雄性具有 异常的生殖行为因此，该补助金旨在确定SCN中Six 6的必要性， 昼夜节律和生殖，并在机械水平上检查Six 6和Six 3如何调节SCN 功能第一个目标将检查在神经元移植后SCN中有条件地缺失Six 6的小鼠。 分化以确定成人SCN中的Six 6是否是正常昼夜节律所必需的。这些实验 将使用转轮运行活动的行为分析，使用Per 2：：荧光素酶的三重转基因小鼠， 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、Per 2和Bmal 1 mRNA的表达。第二个目标将使用这些老鼠来确定， SCN中的Six 6对于正常的生育力、正常的发情周期以及昼夜节律的发生是必要的。 调节LH激增。第三个目的是检验Six 6和Six 3是直接转录因子的假设， Avp、Vip、Per 2和Bmal 1。这些实验将使用瞬时转染和定点突变 测试Six 6和Six 3在昼夜节律和SCN调节中的充分性和必要性的研究 神经肽基因本研究将通过检测新基因阐明SCN的功能机制 候选人，Six 6和Six 3，在昼夜节律和生殖生物学。这些目标将改善 了解昼夜节律和不孕症。