The Kiss1 system in the neuroendocrine control of reproduction

描述（由申请人提供）：这份K99/R00提案旨在补充和推进我之前的研究经验。简而言之，我的专长是研究调节生殖功能的神经机制。我对kisspeptin（由Kiss1基因编码）作为促性腺激素释放激素（GnRH）释放的调节通道的作用进行了表征。在我的博士后研究期间，我扩大了这一研究范围，包括kisspeptin释放的中枢调节剂，神经激肽B （NKB）和动力啡肽A (Dyn)，它们在弓状核（ARC）中与kisspeptin共表达。我们提出了一个模型，其中NKB和Dyn通过反复的附属体来塑造kisspeptin的脉动释放，最终导致GnRH释放的脉冲。本研究计划的总体目标是了解调节GnRH分泌和繁殖的Kiss1神经元亚群的生理功能。表达Kiss1的神经元存在于下丘脑的ARC和前腹侧心室周围核（AVPV）两个离散的核中，分别是雌二醇E2对GnRH分泌的负反馈和正反馈作用的直接靶点。Kiss1在这些神经元中的表达在青春期开始时增加，人类和啮齿动物携带该基因或其受体（Kiss1r）失活突变，表现出青春期发育和不育。本研究的目的是利用遗传小鼠模型结合病毒依赖基因治疗来解剖ARC和AVPV的Kiss1和NKB神经元群在控制繁殖中的差异作用。我们将使用在Kiss1启动子下表达Cre重组酶和GFP的基因工程小鼠。纯合子将导致Kiss1缺陷（但Cre/GFP阳性）小鼠。使用cre依赖的腺相关病毒构建体，我们的目标是在ARC或AVPV的Kiss1神经元中特异性地重新插入Kiss1基因。该项目的具体目的是：1)确定雄性和雌性小鼠的ARC与AVPV中Kiss1神经元在控制青春期发生中的作用；2)确定Kiss1神经元在性类固醇正负反馈中的作用；3)确定NKB在ARC中对雌雄小鼠青春期发育的控制作用。本提案中概述的研究旨在提高我们对生殖神经内分泌学的基础知识，以制定更好的治疗生殖疾病的策略，并提供改进的避孕方法。完成这些研究将提供以下方面的培训：(a)最先进的遗传和分子方法；(b)通过与临床研究人员的密切互动，在我的研究中增加了翻译成分；(c)作为一名成功的独立首席研究员所必需的技能。这种额外的培训将有助于我发展一个有竞争力的研究项目，并有助于我成为一名独立的研究者。

DESCRIPTION (provided by applicant): This K99/R00 proposal is designed to complement and advance my prior research experience. Briefly, my expertise is in the study of the neuronal mechanisms that modulate reproductive function. I have contributed to the characterization of the roles of kisspeptins (encoded by the Kiss1 gene) as regulatory conduits of gonadotropin-releasing hormone (GnRH) release. During my post-doctoral fellowship, I have broadened this research to include the central modulators of kisspeptin release, neurokinin B (NKB) and dynorphin A (Dyn), which are co-expressed with kisspeptin in the arcuate nucleus (ARC). We have proposed a model whereby NKB and Dyn act through recurrent collaterals to shape the pulsatile release of kisspeptin that would ultimately lead to pulses in GnRH release. The overall goal of this research proposal is to understand the physiological function of subpopulations of Kiss1 neurons that regulate the secretion of GnRH and hence reproduction. Kiss1 expressing neurons are found in two discrete nuclei in the hypothalamus, the ARC and anteroventral periventricular nucleus (AVPV), and are direct targets for the negative and positive feedback action of estradiol E2 on GnRH secretion, respectively. Kiss1 expression in these neurons increases at the time of puberty onset and humans and rodents bearing inactivating mutations in this gene or its receptor (Kiss1r) exhibit impuberism and infertility. The objective of this proposal is to use genetic mouse models in combination with viral-dependent gene therapy to dissect the differential role of the ARC and AVPV Kiss1 and NKB neuronal populations in the control of reproduction. We will use genetically engineered mice that express Cre recombinase and GFP under the Kiss1 promoter. Breeding to homozygosity would lead to Kiss1 deficient (but Cre/GFP positive) mice. Using Cre-dependent adeno-associated viral constructs, we aim to reinsert the Kiss1 gene specifically in Kiss1 neurons of the ARC or the AVPV. The specific aims of the project are to: 1) determine the role of Kiss1 neurons in the ARC vs. AVPV in the control of puberty onset in male and female mice; 2) determine the role of Kiss1 neurons in the ARC vs. AVPV in the positive and negative feedback of sex steroids; and 3) determine the role of NKB in the ARC in the control of puberty in male and female mice. The studies outlined in this proposal are designed to advance our fundamental knowledge of reproductive neuroendocrinology, for the development of better strategies to treat reproductive disorders and offer improved methods of contraception. Completion of these studies will provide training in: (a) state-of-the-art geneti and molecular methodologies; (b) addition of a translational component to my research studies through close interaction with clinical investigators; and (c) skills necessary for success as an independent principal investigator. This additional training will aid in my development of a competitive research program and contribute to my establishment as an independent investigator.