Isolation and Culture of Mouse Ovarian Stem Cells

For most experiments (except as indicated below), OSCs were isolated from ovaries of young adult mice (2–3 months of age) by FACS using a C-terminal DDX4-specific antibody (ab13840, Abcam). The cells were analyzed immediately or established in culture without somatic feeder cells, as described^{16 (link), 17 (link), 46 (link), 93 (link)}. Purified mouse OSCs propagated under these conditions spontaneously differentiate into IVD-oocytes for up to 72 h after passage until confluence is regained, and the number of IVD-oocytes generated by a fixed number of OSCs seeded per well remains relatively constant over successive passages^{16 (link), 17 (link), 19 (link)}. Between passages 32–40, OSCs were transfected with the desired plasmids (pStra8-HSVtk or pStra8-Gfp, each containing a neomycin resistance gene) using Lipofectamine 2000 (Invitrogen) and then selected by G418 (Geneticin, Cellgro) over 2 weeks. Cells were then maintained in G418 for all experiments, and the number of IVD-oocytes generated and released into the medium after treatment with vehicle or GCV (2 μM) was then determined by direct visual counts under a microscope^{16 (link), 17 (link), 19 (link)}. In other experiments, GFP-positive cells in ovaries of pStra8-Gfp transgenic female mice were quantitated and then isolated by FACS for gene expression profiling.