The Kiss1 system in the neuroendocrine control of reproduction

描述(申请人提供)：这份K99/R00建议书旨在补充和提升我以前的研究经验。简而言之，我的专长是研究调节生殖功能的神经机制。我参与了Kispeptins(由Kiss1基因编码)作为促性腺激素释放激素(GnRH)释放调节通道的作用的研究。在我的博士后研究期间，我扩大了这项研究的范围，包括Kispeptin释放的中枢调节剂，神经激肽B(NKB)和强啡肽A(Dyn)，它们与Kispeptin在弓状核(ARC)中共同表达。我们已经提出了一个模型，在这个模型中，NKB和Dyn通过反复的侧支作用，塑造Kispeptin的脉动性释放，最终导致GnRH释放的脉冲。这项研究的总体目标是了解Kiss1神经元亚群的生理功能，这些神经元亚群调节GnRH的分泌，从而调节生殖。Kiss1表达神经元分布于下丘脑ARC和室周前核(AVPV)两个不同的核中，分别是雌二醇对GnRH分泌的负反馈和正反馈作用的直接靶点。这些神经元中Kiss1的表达在青春期开始时增加，携带该基因或其受体(Kiss1r)失活突变的人和啮齿动物表现出青春期未成熟和不育。这项建议的目的是使用遗传小鼠模型结合病毒依赖的基因治疗来剖析ARC和AVPV Kiss1和NKB神经元群体在控制生殖方面的不同作用。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。纯合子繁殖会导致Kiss1缺陷(但Cre/GFP阳性)小鼠。利用依赖Cre的腺相关病毒载体，我们的目标是将Kiss1基因特异性地重新插入ARC或AVPV的Kiss1神经元中。该项目的具体目标是：1)确定ARC和AVPV中Kiss1神经元在控制雄性和雌性小鼠青春期开始中的作用；2)确定ARC和AVPV中Kiss1神经元在性类固醇正反馈和负反馈中的作用；以及3)确定NKB在ARC中在控制雄性和雌性小鼠青春期中的作用。这项建议中概述的研究旨在增进我们对生殖神经内分泌学的基础知识，以便制定更好的策略来治疗生殖障碍，并提供改进的避孕方法。这些研究的完成将在以下方面提供培训：(A)最先进的Geneti和分子方法学；(B)通过与临床研究人员的密切互动，在我的研究研究中增加翻译部分；以及(C)作为独立首席研究员取得成功所需的技能。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调查员。

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.