Kisspeptin/Kiss1r in the Female Neuroendocrine Axis

描述（由申请人提供）：排卵是由大脑中的神经回路触发的，大脑感觉到雌二醇（E2）的上升趋势，并在适当的时间产生促性腺激素释放激素（GnRH）和促黄体激素（LH）的激增，引起排卵。然而，大脑中协调这种现象的细胞和分子通路仅被部分理解。在啮齿类动物中，前腹侧室周核（AVPV）包括产生GnRH/LH峰所需的部分电路;然而，直到最近，AVPV内服务于此功能的神经元的表型仍是一个谜。在过去的3年中，它已成为广泛接受的Kiss 1基因的产物，kisspeptin，提供了一个重要的，也许是必要的信号，GnRH神经元。本提案的总体目标是确定AVPV中Kiss 1神经元在GnRH/LH峰产生中的作用，并揭示该过程中涉及的神经，激素和分子途径。第一个具体目标是确定AVPV中的Kiss 1神经元和由这些特定神经元产生的kispeptin是否对产生GnRH/LH峰至关重要，并描述这些神经元的生物物理特性作为峰的函数。孕激素受体（PR）信号传导是浪涌机制的重要组成部分，但PR在大脑中作用的细胞和分子基础，因为它涉及浪涌是未知的。第二个具体目标是确定PR在AVPV Kiss 1神经元中的功能意义。第三个具体目标是确定控制AVPV中Kiss 1神经元的神经传入和信号通路，并评估其在GnRH/LH分泌背景下的生理意义。实验方法结合了更传统的方法，如原位杂交，免疫组织化学，激素操作和测量，以及创新的基因靶向策略。这些方法包括：1）用GFP和tdTomato鉴定Kiss 1神经元，用于在切片制备中记录; 2）通过使用选择性白喉毒素受体表达来消融特定的神经元; 3）通过将荧光标记的伪狂犬病病毒引入Kiss 1神经元，用逆行追踪来映射传入输入到Kiss 1神经元; 4）使用慢病毒递送系统敲低和敲入Kiss 1神经元中的特异性基因;以及5）通过收获单个细胞并采用新的“核糖标记”方法来对Kiss 1神经元的转录组进行指纹分析。在这个建议中描述的研究利用多学科的方法来推进我们对女性生殖生命周期中的一个关键因素--控制排卵的神经内分泌机制的理解。 公共卫生相关性：相关性阐明大脑如何触发导致排卵的神经内分泌事件可以为理解某些女性生殖障碍提供知识平台，包括延迟或性早熟和月经周期功能障碍，并可能指导我们改进治疗方法。这一知识也可以作为基础，为激素避孕和不孕症治疗的更新和更好的战略发展，从而提供希望和选择的妇女和他们的亲人谁是从事计划生育。

DESCRIPTION (provided by applicant): Ovulation is triggered by neural circuits in the brain, which senses a rising tide of estradiol (E2) and-at the right time-generate a surge of gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH), causing ovulation. However, the cellular and molecular pathways in the brain that orchestrate this phenomenon are only partially understood. In rodent species, the anteroventral periventricular nucleus (AVPV) comprises part of the circuitry necessary to produce the GnRH/LH surge; however, until recently, the phenotype of the neurons within the AVPV that serve this function were a mystery. Within the past 3 years, it has become widely accepted that a product of the Kiss1 gene, kisspeptin, provides an important-perhaps essential-signal to GnRH neurons. The overall goal of this proposal is to identify the role that Kiss1 neurons in the AVPV play in the generation of the GnRH/LH surge and to reveal the neural, hormonal, and molecular pathways involved in that process. The first specific aim is to determine whether Kiss1 neurons in the AVPV and kispeptin produced by those particular neurons are essential for generating the GnRH/LH surge and to delineate the biophysical properties of those neurons as a function of the surge. Progesterone receptor (PR) signaling is an essential component of the surge mechanism, but the cellular and molecular basis of PR's action in the brain as it relates to the surge is not known. The second specific aim is designed to determine the functional significance of PR in Kiss1 neurons of the AVPV. The third specific aim is to identify the neural afferents and signaling pathways that control Kiss1 neurons in the AVPV and to evaluate their physiological significance in the context of GnRH/LH secretion. The experimental approach combines more traditional methodologies, such as in situ hybridization, immunohistochemistry, and hormone manipulations and measurements, together with innovative gene-targeting strategies. These include methods to 1) identify Kiss1 neurons with GFP and tdTomato for recording in slice preparations; 2) ablate specific neurons through the use of selective diphtheria toxin receptor expression; 3) map the afferent inputs to Kiss1 neurons with retrograde tracing by introducing a fluorescent-tagged pseudorabies virus into Kiss1 neurons; 4) knock-down and knock-in specific genes in Kiss1 neurons with the use of a lentivirus delivery system; and 5) fingerprint the transcriptome of Kiss1 neurons by harvesting individual cells and employing a new "ribotagging" methodology. The studies described in this proposal utilize a multi-disciplinary approach to advance our understanding of a critical element in the female reproductive life cycle-the neuroendocrine mechanism that governs ovulation. PUBLIC HEALTH RELEVANCE: RELEVANCE Elucidating how the brain triggers the neuroendocrine events that lead to ovulation could provide the intellectual platform for understanding certain disorders of female reproduction, including delayed or precocious puberty and menstrual cycle dysfunction-and perhaps guide us toward improved therapies for their treatment. This knowledge could also serve as the basis for the development of newer and better strategies for hormonal contraception and infertility treatment-thus, offering hope and freedom of choice to women and their loved ones who are engaged in family planning.