Neural Control of the Prepubertal Ovary

描述（由申请人提供）：我们对控制卵巢发育的激素机制了解甚多。最近，该领域的一个主要关注焦点是确定在卵巢微环境中运作的调节途径，这些途径有助于获得卵巢生殖能力。在这个框架内，我们的实验室已经发展了神经营养因子（nt）及其Trk酪氨酸激酶受体的概念，长期以来被认为是神经系统发育所必需的，也参与了卵巢功能的控制。利用基因靶向方法，我们发现trkB是神经营养因子4/5 （NT-4/5）和脑源性神经营养因子（BDNF）的高亲和力受体，是早期卵泡生长和卵母细胞存活所需的信号分子。此外，我们发现神经生长因子（NGF）对卵泡生长的启动有独立的贡献。其他研究表明，NGF通过trkA受体对排卵也很重要，但尽管有这种生理作用，但卵巢内NGF合成的不适当持续增加会导致功能改变，从而导致卵泡囊肿的发生。基于这些发现，本更新申请提出以下具体目标：1)确定早期卵泡生长和卵母细胞存活所需的TrkB受体亚型（全长或截短），并鉴定主要响应TrkB信号的细胞。利用Cre-loxP技术特异性地破坏卵母细胞或颗粒细胞中全长和截断的TrkB亚型的表达，将实现本Aim的目标。2)验证通过TrkB受体介导的nt信号通过支持卵母细胞-颗粒细胞Jagged 1-Notch2通讯通路促进卵泡早期生长的假设。这一目标将通过结合使用细胞特异性trkB KOs和细胞/生化方法来定义trkB信号和Notch2通路之间存在的关系来实现。3)为了验证NGF依赖性trkA信号是卵泡正常发育和排卵所必需的假设，NGF的过量产生损害了卵泡进入排卵前阶段的能力，从而建立了导致多囊卵巢发育的条件。为了实现这一目标，我们将使用以细胞特异性方式过度表达NGF的转基因小鼠，以及通过p75（常见的NT受体）或trkA（高亲和的NGF受体）在卵巢细胞中有条件地破坏信号的小鼠。4)验证卵巢NGF过剩在局部微环境中创造有利于非人灵长类动物多囊卵巢发育的条件的假设。为了实现这一目标，我们将使用慢病毒传递系统来增强成年非人灵长类动物卵巢间质室中神经生长因子的产生。

DESCRIPTION (provided by applicant): Much is known about the hormonal mechanisms controlling ovarian development. More recently, a major focus of attention in the field has been the identification of regulatory pathways that, operating within the ovarian microenvironment, contribute to the acquisition of ovarian reproductive competence. Within this framework, our laboratory has developed the concept that neurotrophins (NTs) and their Trk tyrosine kinase receptors, long thought to be exclusively required for the development of the nervous system are also involved in the control of ovarian function. Employing gene targeting approaches we identified trkB, the high-affinity receptor for neurotrophin-4/5 (NT-4/5) and brain-derived neurotrophic factor (BDNF), as a signaling molecule required for early follicular growth and oocyte survival. In addition, we showed that nerve growth factor (NGF) contributes independently to the initiation of follicular growth. Other studies indicated that NGF acting via trkA receptors is also important for ovulation, but that despite this physiological role, an inappropriately sustained increase in intraovarian NGF synthesis results in functional alterations leading to the development of follicular cysts. Based on these findings, the present renewal application proposes the following Specific Aims: 1) To define the TrkB receptor isoform (full-length or truncated) required for early follicle growth and oocyte survival, and identify the cells primarily responsive to TrkB signaling. The objectives of this Aim will be achieved using Cre-loxP technology to specifically disrupt the expression of full-length and truncated TrkB isoforms in either oocytes or granulosa cells. 2) To test the hypothesis that NTs signaling via TrkB receptors promote early follicular growth by supporting an oocyte-to granulosa cell Jagged 1-Notch2 communication pathway. This aim will be achieved with the combined use of cell-specific trkB KOs and cellular/biochemical approaches to define the relationship that exists between TrkB signaling and the Notch2 pathway. 3) To test the hypotheses that while NGF-dependent trkA signaling is required for the normal development of antral follicles and ovulation, an overproduction of NGF compromises the ability of antral follicles to reach a preovulatory stage, and thus establishes conditions leading to the development of polycystic ovaries. To accomplish this Aim we will use transgenic mice that overexpress NGF in a cell specific manner, and mice in which signaling through p75 (the common NT receptor), or trkA (the high-affinity NGF receptor) are conditionally disrupted in ovarian cells. 4) To test the hypothesis that an excess of ovarian NGF creates conditions in the local microenvironment that favor the development of polycystic ovaries in nonhuman primates. To accomplish this Aim we will use a lentiviral delivery system to enhance the production of NGF in the interstitial compartment of the adult nonhuman primate ovary.