Oocyte formation by mitotically active germ cells purified from ovaries of reproductive-age women

Abstract

In 2004, a team led by Jonathan Tilly reported that mice contained oogonial stem cells (OSCs), suggesting that females may be able to generate new oocytes in adulthood—a concept that was, and still is, quite controversial even though those findings have since been replicated by others. In a new report, Tilly and colleagues now perfect the purification of mouse OSCs and, using this technique, they show that similar cells exist in women of reproductive age. They also show that the mouse and human OSCs are able to give rise to oocytes in vivo (in the case of the human cells after xenotransplantation into NOD-SCID mice), while also showing that the mouse OSCs can give rise to embryos after in vitro fertilization. Germline stem cells that produce oocytes in vitro and fertilization-competent eggs in vivo have been identified in and isolated from adult mouse ovaries. Here we describe and validate a fluorescence-activated cell sorting-based protocol that can be used with adult mouse ovaries and human ovarian cortical tissue to purify rare mitotically active cells that have a gene expression profile that is consistent with primitive germ cells. Once established in vitro, these cells can be expanded for months and can spontaneously generate 35- to 50-μm oocytes, as determined by morphology, gene expression and haploid (1n) status. Injection of the human germline cells, engineered to stably express GFP, into human ovarian cortical biopsies leads to formation of follicles containing GFP-positive oocytes 1–2 weeks after xenotransplantation into immunodeficient female mice. Thus, ovaries of reproductive-age women, similar to adult mice, possess rare mitotically active germ cells that can be propagated in vitro as well as generate oocytes in vitro and in vivo.