Kisspeptin/Kiss1r in the Female Neuroendocrine Axis

描述(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)和黄体生成素(LH)的激增，导致排卵。然而，人们对大脑中协调这一现象的细胞和分子通路只有部分了解。在啮齿动物中，前腹侧脑室周围核(AVPV)组成了产生GnRH/LH峰所必需的电路的一部分；然而，直到最近，AVPV内服务于这一功能的神经元的表型仍然是一个谜。在过去的三年里，人们普遍认为Kiss1基因的产物Kispeptin为GnRH神经元提供了一个重要的--也许是必要的--信号。这项建议的总体目标是确定AVPV中的Kiss1神经元在GnRH/LH峰的产生中所起的作用，并揭示参与这一过程的神经、激素和分子通路。第一个特定的目的是确定AVPV中的Kiss1神经元和由这些神经元产生的KiPeptin是否对GnRH/LH峰的产生是必要的，并描绘这些神经元的生物物理特性作为该峰的函数。孕激素受体(PR)信号是电涌机制的重要组成部分，但PR在脑内与电涌相关的作用的细胞和分子基础尚不清楚。第二个特定目的是确定PR在AVPV的Kiss1神经元中的功能意义。第三个具体目的是确定控制动静脉动静脉内Kiss1神经元的神经传入和信号通路，并在促性腺激素释放激素/促黄体生成素分泌的背景下评价它们的生理学意义。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Kiss1神经元，以便在切片标本中记录；2)通过选择性白喉毒素受体表达来消融特定神经元；3)通过将荧光标记的伪狂犬病病毒导入Kiss1神经元进行逆行追踪，将传入输入映射到Kiss1神经元；4)通过慢病毒传递系统在Kiss1神经元中下调和敲入特定基因；以及5)通过收集单个细胞和使用新的“Ribottag”方法来指纹Kiss1神经元的转录组。这项建议中描述的研究利用多学科的方法来促进我们对女性生殖生命周期中的一个关键因素--支配排卵的神经内分泌机制--的理解。 公共卫生相关性：阐明大脑如何触发导致排卵的神经内分泌事件，可以为理解女性生殖的某些障碍提供智力平台，包括青春期延迟或性早熟和月经周期障碍--或许还能指导我们改进治疗方法。这一知识还可以作为制定新的和更好的荷尔蒙避孕和不孕不育治疗战略的基础，从而为从事计划生育的妇女及其亲人提供希望和选择自由。

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.