Neural Control of the Prepubertal Ovary

描述(由申请人提供)：对控制卵巢发育的荷尔蒙机制有很多了解。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行的有助于获得卵巢生殖能力的调控途径。在这个框架内，我们的实验室提出了神经营养素(NTS)及其Trk酪氨酸激酶受体的概念，长期以来，神经营养素(NTS)及其Trk酪氨酸激酶受体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。通过基因打靶的方法，我们发现神经营养素-4/5(NT-4/5)和脑源性神经营养因子(BDNF)的高亲和力受体TrkB是早期卵泡生长和卵母细胞存活所必需的信号分子。此外，我们还发现神经生长因子(NGF)在卵泡生长的启动中起着独立的作用。其他研究表明，NGF通过TrkA受体发挥作用对排卵也很重要，但尽管有这种生理作用，卵巢内NGF合成的不适当持续增加会导致功能改变，导致卵泡囊肿的发展。基于这些发现，目前的更新应用提出了以下具体目标：1)定义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)，并鉴定主要对TrkB信号做出反应的细胞。这一目标的实现将使用Cre-loxP技术来特异性地干扰全长和截短的TrkB亚型在卵母细胞或颗粒细胞中的表达。2)验证NTS信号通过TrkB受体支持卵母细胞-颗粒细胞交错的1-Notch2通讯通路促进早期卵泡生长的假说。这一目标将通过结合使用细胞特异性TrkB KO和细胞/生化方法来确定TrkB信号和Notch2途径之间存在的关系来实现。3)验证如下假设：尽管有腔卵泡的正常发育和排卵需要依赖于NGF的TrkA信号，但过量的NGF会损害有腔卵泡达到排卵前的能力，从而为多囊卵巢的发育创造条件。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(高亲和力NGF受体)在卵巢细胞中有条件地中断信号的小鼠。4)验证卵巢神经生长因子过量在局部微环境中为非人类灵长类动物多囊卵巢发育创造条件的假设。为了实现这一目标，我们将使用慢病毒递送系统来增强成年非人灵长类动物卵巢间质中NGF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.