Reversible Contraception by Selective Silencing of GnRH-II

项目摘要 这项提案的总体目标是提升我们对灵长类动物神经内分泌生殖系统的理解。 轴，从而为新型避孕药的开发奠定基础，并获得新的见解 关于人类生殖系统疾病的病因学因此，这一目标是响应NICHD的一个 新的研究重点：“鼓励创新技术和模型系统的发展， 研究生育和不孕症。”具体来说，拟议的研究将实证检验的假设，积极的 抗GnRH-II免疫可提供安全有效的长期避孕--通过选择性沉默 LH激增机制，同时保持神经内分泌生殖轴的其余部分完好无损。 在人类中，促性腺激素释放激素（GnRH）神经元代表了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，人类和恒河猴（但不是啮齿动物）被发现 表达两种不同分子形式的GnRH（GnRH-I和GnRH-II）。此外，只有GnRH-II 神经元被发现对雌激素反馈有积极的反应，这表明GnRH-II神经元作为雌激素受体， 周期中期排卵前LH峰的主要触发因素。因此，我们假设选择性沉默 GnRH-II神经元将阻断排卵而不干扰卵巢类固醇生成或影响负反馈 促性腺激素释放激素I型神经元上的雌激素。这一假设将使用雌性恒河猴， 高度转化的非人灵长类动物模型，其显示类似人类的月经周期和类似的 组织的生殖神经内分泌轴。具体目标1：检验活性 针对GnRH-II的免疫将选择性地阻断排卵而不影响卵巢类固醇生成。我们 将通过免疫成年雌性恒河猴来测试这一假设， GnRH-II神经肽;对照动物将针对GnRH的独特非保守区进行免疫， I.免疫接种后，每天检查动物的月经体征和循环水平， 生殖激素（即，LH、FSH、雌二醇、孕酮、抗苗勒管激素）将在双 每周血液样本。我们希望表明，选择性免疫GnRH-II将阻止发展 周期中期LH激增和黄体形成（即，由维持低循环推断 孕酮水平）。具体目标2：阐明神经内分泌机制， GnRH-II沉默的避孕潜力。使用远程连续血液采样，我们将检查动态 免疫后循环中生殖激素水平之间的关系（1）卵巢完整 动物，（2）卵巢切除术后，和（3）雌激素诱导LH峰后。我们希望证明选择性的 GnRH-II的沉默将完全阻断神经内分泌系统的正性雌激素反馈成分， 生殖轴，而离开GnRH-I介导的负反馈组件功能完整。

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.