Lineage tracing of germline stem cell differentiation in adult ovaries

描述(申请人提供)：几十年来，人们相信哺乳动物雌性在胚胎发育过程中依靠原始生殖细胞(PGC)来产生全部的卵母细胞，导致在出生时获得一个不可再生的含卵母细胞的卵泡池。最近在成年哺乳动物卵巢中发现的雌性生殖系干细胞(GSCs)或卵原干细胞(OSCs)开启了卵母细胞池在成年期由OSCs主动补充的前景，在一定程度上类似于由精原干细胞(SSCs)主动维持精子发生。虽然已经有几项研究分离了小鼠的OSCs，移植研究表明OSCs会产生卵子，产生可存活的后代，但OSCs是否会分化为在正常生理条件下成熟和受精的卵母细胞仍有待确定。目前的研究试图通过使用体内细胞谱系追踪来解决这一知识差距，这是表征干细胞功能和检查干细胞维持组织动态平衡的组织谱系发育的有力手段。就我们的目的而言，这项技术将被用来通过永久标记OSCs的即时分化后代来检测OSCs在生理条件下是否经历卵源分化并形成卵母细胞。此外，由于示踪报告基因的激活是在基因组水平上进行的，因此受精后来自标记卵母细胞的后代应该表现出报告基因的整体和结构性表达。为了实现这一目标，我们提出了以下具体目标：1)通过永久标记OSC子代进行减数分裂并跟踪其在体内的生理命运，确定OSC是否主动分化为卵母细胞；以及2)确定OSC来源的卵母细胞在出生后的生活中是否成熟、排卵、受精并在正常生理条件下产生可存活的后代。 成功完成拟议的研究对于我们理解卵巢生物学、女性生殖适合性和不孕不育的发病机制具有直接和基本的重要性，这些都代表着越来越多的公共卫生相关性。

DESCRIPTION (provided by applicant): For decades it was believed that mammalian females rely on primordial germ cells (PGCs) to generate their entire quota of oocytes during embryogenesis, leading to endowment of a non-renewable pool of oocyte-containing follicles at birth. The recent discovery of female germline stem cells (GSCs) or oogonial stem cells (OSCs) in adult mammalian ovaries opens the prospects that the oocyte pool is actively replenished during adulthood by OSCs, paralleling to a degree the active maintenance of spermatogenesis by spermatogonial stem cells (SSCs). Although several studies have isolated mouse OSCs and shown by transplantation studies that OSCs generate eggs which produce viable offspring, it remains to be determined if OSCs differentiate into oocytes that mature and fertilize under normal physiological conditions. The present study seeks to address this gap in knowledge by using in-vivo cell lineage tracing, which is a powerful means to characterize stem cell function and examine lineage development in tissues where stem cells maintain tissue homeostasis. For our purposes, this technology will be used to examine whether OSCs undergo oogenic differentiation and form oocytes under physiological conditions by permanently marking the immediate differentiating progeny of OSCs. Moreover, since activation of the tracing reporter occurs at a genomic level, the offspring potentially derived from "marked" oocytes after fertilization should exhibit global and constitutive expression of the reporter gene. To achieve this, we propose the following Specific Aims: 1) determine if OSCs actively differentiate into oocytes by permanently marking OSC progeny committing to meiosis and following their physiological fate in vivo; and, 2) determine if OSC-derived oocytes generated during postnatal life mature, ovulate, fertilize and produce viable offspring under normal physiological conditions. Successful completion of the proposed studies bears immediate and fundamental importance to our understanding of ovarian biology, female reproductive fitness and the pathogenesis of infertility, which represent increasing public health relevance.