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在胚胎发生过程中依赖原始生殖细胞（PGCs）产生其全部卵母细胞配额，导致出生时提供不可再生的含有卵母细胞的卵泡池。最近在成年哺乳动物卵巢中发现的雌性种系干细胞（GSCs）或卵母干细胞（OSCs）为卵母细胞池在成年期由OSCs积极补充开辟了前景，在一定程度上平行于精原干细胞（ssc）积极维持精子发生。虽然有几项研究已经分离出小鼠OSCs，并通过移植研究表明，OSCs产生的卵子能够产生可存活的后代，但OSCs是否分化为卵母细胞，并在正常生理条件下成熟和受精，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空白，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。就我们的目的而言，该技术将用于通过永久标记osc的直接分化后代来检查osc是否在生理条件下进行卵源分化并形成卵母细胞。此外，由于追踪报告基因的激活发生在基因组水平，因此受精后可能来自“标记”卵母细胞的后代应该表现出报告基因的全局和组成性表达。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后代进行减数分裂并遵循其体内生理命运来确定OSC是否主动分化为卵母细胞；2)在正常生理条件下确定出生后产生的osc来源的卵母细胞是否成熟、排卵、受精并产生可存活的后代。

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. PUBLIC HEALTH RELEVANCE: Aging and pathological conditions cause exhaustion of the oocyte reserve and ovarian failure. Stem cell- based maintenance of ovarian function may provide a novel means to achieve significant improvements in fertility, health and well-being, all of which represent significant public health relevance.