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.