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，它感觉到雌二醇（E2）的上升，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，大脑中协调这一现象的细胞和分子途径只被部分理解。在啮齿类动物中，腹前侧心室周围核（AVPV）包含产生GnRH/LH激增所需的部分电路；然而，直到最近，AVPV中发挥这一功能的神经元的表型还是一个谜。在过去的3年里，Kiss1基因的产物kisspeptin为GnRH神经元提供了一个重要的——也许是必要的——信号，这一观点已被广泛接受。本提案的总体目标是确定AVPV中的Kiss1神经元在GnRH/LH激增产生中的作用，并揭示参与该过程的神经、激素和分子途径。第一个具体目标是确定AVPV中的Kiss1神经元和这些特定神经元产生的kispeptin是否对产生GnRH/LH激增至关重要，并描述这些神经元的生物物理特性作为激增的功能。孕激素受体（PR）信号是高潮机制的重要组成部分，但PR在大脑中与高潮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。第三个具体目标是确定控制AVPV中Kiss1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Kiss1神经元，以便在切片制备中记录；2)通过选择性白喉毒素受体表达，消融特定神经元；3)通过将荧光标记的伪狂犬病毒引入Kiss1神经元，逆行示踪，将传入输入映射到Kiss1神经元；4)使用慢病毒传递系统敲低和敲入Kiss1神经元中的特定基因；5)通过收集单个细胞并采用新的“核糖标记”方法，对Kiss1神经元的转录组进行指纹鉴定。本提案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一个关键因素的理解——控制排卵的神经内分泌机制。

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.