Lineage tracing of germline stem cell differentiation in adult ovaries

描述（由申请人提供）：几十年来，人们认为哺乳动物雌性依赖原始生殖细胞（PGC）在胚胎发生期间产生其全部配额的卵母细胞，导致出生时具有不可再生的含卵母细胞卵泡池。最近在成年哺乳动物卵巢中发现的雌性生殖系干细胞（GSC）或卵原干细胞（OSC）开辟了这样的前景，即卵母细胞池在成年期由OSC积极补充，在一定程度上平行于精原干细胞（SSCs）积极维持精子发生。尽管一些研究已经分离出小鼠OSCs，并通过移植研究表明OSCs产生能产生存活后代的卵子，但OSCs是否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。本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稳态的组织中谱系发育的有力手段。为了我们的目的，该技术将用于检查OSC是否经历卵细胞分化和形成卵母细胞在生理条件下通过永久标记的直接分化的后代的OSC。此外，由于示踪报告基因的激活发生在基因组水平，受精后可能来源于“标记”卵母细胞的后代应表现出报告基因的整体和组成型表达。为了实现这一点，我们提出了以下具体目的：1）通过永久标记进行减数分裂的OSC后代并跟踪其体内生理命运来确定OSC是否主动分化成卵母细胞;以及2）确定在出生后生命期间产生的OSC衍生的卵母细胞是否在正常生理条件下成熟、排卵、受精并产生存活后代。 成功完成这些研究对我们理解卵巢生物学、女性生殖健康和不孕症的发病机制具有直接和根本的重要性，这些研究代表着越来越多的

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