Role of neuroestrogens in control of GnRH release

描述（由申请人提供）：本提案的总体目标是研究非人灵长类动物促性腺激素释放激素（GnRH）神经元的调节。GnRH从下丘脑释放，通过垂体促性腺激素控制生殖功能。拟议的研究旨在增加我们对雌二醇（E2）调节GnRH释放的理解。最近，E2在大脑中合成和释放的概念在学习/记忆以及性行为中起着重要作用。虽然众所周知，卵巢循环E2通过负反馈和正反馈机制调节下丘脑-垂体功能，但我们通过微透析方法的初步数据表明，下丘脑合成和释放的E2似乎在调节GnRH释放中发挥作用。即：1)在体内将雌二醇-苯甲酸酯（estradiol benzoate， EB）短暂注入卵巢切除（OVX）雌性猴的茎中突（S-ME）刺激GnRH的释放，且潜伏期短（<10 min）； 2)这种快速的GnRH释放伴随着E2的释放；3)电刺激下丘脑内侧基底部也刺激GnRH和E2的释放；4)芳香化酶抑制剂来曲唑抑制GnRH和E2的自发和EB诱导释放。值得注意的是，在微透析液中发现的E2来源必须是下丘脑起源，因为猴子是OVX，并且用液相色谱-质谱（LC/MS/MS）方法将E2与EB区分开来。因此，在这个R21提案中，我们将测试神经雌激素对GnRH释放的控制很重要的假设，因此生殖功能。Aim 1将验证E2对GnRH释放的快速作用代表下丘脑中神经雌二醇的作用，S-ME中神经雌二醇的释放有助于调节GnRH/LH释放的假设。这将通过检查EB、ES和来曲唑对GnRH/LH释放的影响来完成。目的2将检验EB暴露期对GnRH/LH释放的快速刺激作用与负反馈抑制E2作用的区别。实验将在OVX成年雌性猴子中进行，采用体内微透析方法和连续血液采样。透析液中的GnRH和血浆样品中的LH将通过RIA和透析液中的E2水平通过LC/MS/MS进行评估。在非人类灵长类动物中拟议项目的结果将为开发R01项目提供基础，从而导致神经雌激素参与控制GnRH释放的开创性研究。PI相信，这将最终为人类生殖功能的神经内分泌机制带来新的见解。

DESCRIPTION (provided by applicant): The overall objective of this proposal is to investigate the regulation of gonadotropin-releasing hormone (GnRH) neurons in the non-human primate. GnRH is released from the hypothalamus and controls reproductive function through pituitary gonadotrophins. The proposed studies are designed to increase our understanding of estradiol (E2) regulation of GnRH release. Recently, the concept that E2 synthesized and released in the brain plays an important role in learning/memory as well as sexual behaviors has emerged. Although it is well known that circulating E2 from the ovary regulates hypothalamo-pituitray function via negative and positive feedback mechanisms, our preliminary data with a microdialysis method indicate that E2 synthesized and released in the hypothalamus appears to play a role in the regulation of GnRH release. That is, 1) a brief infusion of estradiol benzoate (EB) into the stalk-median eminence (S-ME) in vivo in ovariectomized (OVX) female monkeys stimulated GnRH release with a short latency (<10 min), 2) this rapid GnRH release is accompanied by E2 release, 3) electrical stimulation (ES) of the medial basal hypothalamus also stimulated both GnRH and E2 release, and 4) the aromatase inhibitor, letrozole, suppressed spontaneous and the EB-induced release of GnRH and E2. Remarkably, the source of E2 found in microdialysates must be of hypothalamic origin, as monkeys were OVX and analysis with a liquid chromatography-mass spectrometry (LC/MS/MS) method distinguishes E2 from EB. Therefore, in this R21 proposal we will test the hypothesis that neuroestrogens are important for control of GnRH release, hence reproductive function. Aim 1 will test the hypothesis that the rapid action of E2 on GnRH release represents neuroestradiol action in the hypothalamus and neuroestradiol release in the S-ME contributes to regulation of GnRH/LH release. This will be accomplished by examining the effects of EB, ES, and letrozole on GnRH/LH release. Aim 2 will test the hypothesis that the EB exposure period distingushes rapid stimulatory action from negative feedback inhibitory E2 action on GnRH/LH release. Experiments will be conducted in OVX adult female monkeys using an in vivo microdialysis method and serial blood sampling. GnRH in dialysates and LH in plasma samples will be assessed by RIA and E2 levels in dialysates by LC/MS/MS. Results from the proposed project in non-human primates will provide a basis for developing R01 projects leading to groundbreaking studies of the involvement of neuroestrogens in control of GnRH release. The PI believes that this will ultimately bring new insights into the neuroendocrine mechanism of reproductive function in humans.