Germ Cell Specification from Murine Embryonic Stem Cells: A Model for the Identification of Irreversible Meiotic Commitment.

Abstract

Despite evidence indicating that differentiating mouse embryonic stem cells (mESC) can generate oocytes and follicle-like structures in vitro, fertilizable oocytes have not been produced. Unlike endogenous primordial germ cells (PGC), which have the innate ability to form oocytes that initiate and progress through meiosis, mESC-derived germ cells exhibit many meiotic abnormalities and fail to progress through prophase. As such, isolation of early stage (pre-meiotic) ESC-derived germ cells may prove useful for identifying key processes that lead to meiotic commitment and progression. Although the timing of meiotic entry in embryonic ovaries in vivo has been well characterized, the molecular events associated with irreversible meiotic commitment are not well defined. Induction of stimulated by retinoic acid gene 8 (Stra8) is associated with the early onset of meiotic commitment, and has been previously used to isolate male germ cells from mESC cultures. In the present study, we combined a dual reporter system with germline specification by mESC to identify two distinct populations of Stra8-positive germ cells that differ in their degree of commitment to meiosis. To track germ cells, we used a karyotypic XX mESC line derived in our lab from transgenic mice expressing green fluorescent protein (GFP) driven by a fragment of the mouse Oct4 promoter in which the epiblast-specific proximal enhancer has been inactivated (TgOG2 or ΔPE-Oct4-Gfp). When undifferentiated, TgOG2 mESC are GFP positive (GFP+). Following initiation of differentiation by removal of leukemia inhibitory factor (LIF) and mouse embryonic fibroblast (MEF) feeder cells, TgOG2 mESC were transiently transfected with a DsRed2 expression vector under control of the murine Stra8 promoter. In the continued absence of LIF and MEF, the level of GFP gradually decreased such that comparatively few GFP+ cells remained detectable after 5 days; however, DsRed2-expressing cells became identifiable by fluorescence microscopy 5 days after the induction of differentiation. Subsequent isolation by fluorescence-activated cell sorting (FACS) revealed two distinct DsRed2-expressing cell populations: one that was GFP+ and one that was GFP-negative (GFP-). The sorted populations were re-plated separately and while the DsRed+/GFP- population remained quiescent, DsRed+/GFP+ germ cells actively proliferated and maintained high levels of GFP expression. These findings indicate that mESC-derived Stra8+/Oct4- germ cells commit to meiosis and lose replicative capacity; however, there is a subset of Stra8- expressing germ cells that continue to express Oct4 and retain high proliferative potential. In light of recent evidence indicating that Stra8 binds to DNA and exhibits transcriptional activity, we hypothesize that irreversible meiotic entry is a transcriptionally defined event regulated by Stra8 in those germ cells that repress Oct4 expression. (Supported by Ruth L. Kirschstein National Research Service Award F32AG034809 to D.C.W, NIH MERIT Award R37-AG012279 to J.L.T, and Vincent Memorial Research Funds). (poster)