Reversible Contraception by Selective Silencing of GnRH-II

项目总结 这项建议的总体目标是提高我们对灵长类神经内分泌生殖的理解。 轴心，从而为开发新型避孕药奠定基础，并获得新的见解 关于特发性人类生殖障碍的病因学。因此，这一目标是对NICHD的一个 新的研究重点：“鼓励创新技术和模式系统的发展，以 研究生育和不孕不育。具体地说，这项拟议的研究将对活跃的假设进行实证检验 针对GnRH-II的免疫可以提供安全有效的长期避孕--通过选择性沉默 黄体生成素激增机制，而保持神经内分泌生殖轴的其余部分不变。 在人类中，促性腺激素释放激素(GnRH)神经元代表着主要的神经内分泌环节 在大脑和生殖轴的其他部分之间，然而这些神经元触发的机制 人们对排卵知之甚少。然而，最近发现了人类和恒河猴(但不是啮齿动物) 表达两种不同的GnRH分子形式(GnRH-I和GnRH-II)。此外，只有GnRH-II 神经元对雌激素反馈的反应是积极的，这表明GnRH-II神经元是 周期中期排卵前促黄体生成素高峰的主要触发因素。因此，我们假设选择性的沉默 GnRH-II神经元将在不干扰卵巢类固醇合成或影响负反馈的情况下阻止排卵 雌激素对GnRH-I神经元的影响。这一假设将用雌性恒河猴进行经验性检验， 高度翻译的非人类灵长类动物模型，显示出类似人类的月经周期和类似的 其生殖神经内分泌轴的组织。具体目标1：检验活跃的假设 针对GnRH-II的免疫将选择性地阻止排卵，而不会影响卵巢类固醇的生成。我们 将通过免疫成年雌性恒河猴来检验这一假设，以对抗 GnRH-II神经肽；对照动物将针对GnRH-II独特的非保守区进行免疫。 I.免疫后，每天将检查动物的月经迹象和血液循环中的 生殖激素(即黄体生成素、卵泡刺激素、雌二醇、孕酮、抗苗勒氏激素)将在两年内进行监测。 每周一次的血样。我们希望证明针对GnRH-II的选择性免疫将阻止发育 黄体形成和黄体形成(即，通过维持低循环而推断 黄体酮水平)。具体目标2：阐明神经内分泌机制 GnRH-II沉默的避孕潜力。使用远程连续采血，我们将检查动态 免疫后血液中生殖激素水平的关系(1)卵巢完整 动物，(2)卵巢切除后，和(3)雌激素诱导的黄体生成素激增。我们希望展示有选择性的 沉默GnRH-II将完全阻断神经内分泌的雌激素正反馈成分 生殖轴，同时保持GnRH-I介导的负反馈成分的功能完整。

PROJECT SUMMARY The overall goal of this proposal is to upgrade our understanding of the primate neuroendocrine reproductive axis, and thereby to lay a foundation for the development of novel contraceptives and to gain new insights about the etiology of idiopathic human reproductive disorders. This goal is thus responsive to one of NICHD's new Research Priorities: “To Encourage the Development of Innovative Technologies and Model Systems to Study Fertility and Infertility.” Specifically, the proposed research will empirically test the hypothesis that active immunization against GnRH-II can provide safe and effective long-term contraception - by selectively silencing the LH surge mechanism, while leaving the rest of the neuroendocrine reproductive axis intact. In humans, gonadotropin-releasing hormone (GnRH) neurons represent the primary neuroendocrine link between the brain and the rest of the reproductive axis, yet the mechanism by which these neurons trigger ovulation is poorly understood. Recently, however, human and rhesus macaques (but not rodents) were found to express two different molecular forms of GnRH (GnRH-I and GnRH-II). Furthermore, only the GnRH-II neurons were found to respond positively to estrogen feedback, suggesting that GnRH-II neurons serve as the primary trigger of the mid-cycle preovulatory LH surge. We therefore hypothesize that selective silencing of GnRH-II neurons will block ovulation without perturbing ovarian steroidogenesis or affecting negative feedback of estrogen on GnRH-I neurons. This hypothesis will be empirically tested using the female rhesus macaque, a highly translational nonhuman primate animal model that shows human-like menstrual cycles and similar organization of its reproductive neuroendocrine axis. Specific Aim 1: To test the hypothesize that active immunization against GnRH-II will selectively block ovulation without impacting ovarian steroidogenesis. We will test this hypothesis by immunizing adult female rhesus macaques against unique non-conserved regions of the GnRH-II neuropeptide; control animals will be immunized against a unique non-conserved region of GnRH- I. After immunization, the animals will be examined daily for signs of menstruation and circulating levels of reproductive hormones (i.e., LH, FSH, estradiol, progesterone, anti-Müllerian hormone) will be monitored in bi- weekly blood samples. We expect to show that selective immunization against GnRH-II will block development of the mid-cycle LH surge and corpus luteum formation (i.e., inferred by maintenance of low circulating progesterone levels). Specific Aim 2: To elucidate the neuroendocrine mechanism that underlies the contraceptive potential of GnRH-II silencing. Using remote serial blood sampling we will examine the dynamic relationships between reproductive hormone levels in the circulation after immunization (1) in ovary-intact animals, (2) after ovariectomy, and (3) after estrogen induction of an LH surge. We expect to show that selective silencing of GnRH-II will completely block the positive estrogen feedback component of the neuroendocrine reproductive axis, while leaving the GnRH-I-mediated negative feedback component functionally intact.