title: Oocyte formation by mitotically active germ cells purified from ovaries of reproductive-age women.
abstract: 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.
authors.affiliation: Vincent Center for Reproductive Biology, Massachusetts General Hospital Vincent Department of Obstetrics and Gynecology, Massachusetts General Hospital, Boston, Massachusetts, USA.
authors.forename: Yvonne A R
authors.initials: YA
authors.lastname: White
authors.forename: Dori C
authors.initials: DC
authors.lastname: Woods
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Takai
authors.forename: Osamu
authors.initials: O
authors.lastname: Ishihara
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Seki
authors.forename: Jonathan L
authors.initials: JL
authors.lastname: Tilly
mesh_terms: D000818:Animals; D001706:Biopsy; D053487:DEAD-box RNA Helicases / Q000235:genetics / Q000378:metabolism; D005260:Female; D005434:Flow Cytometry / Q000379:methods; D020869:Gene Expression Profiling; D005854:Germ Cells / Q000166:cytology* / Q000254:growth & development / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006801:Humans; D051379:Mice; D008938:Mitosis / Q000235:genetics*; D009865:Oocytes / Q000166:cytology* / Q000254:growth & development*; D006080:Ovarian Follicle / Q000254:growth & development / Q000378:metabolism; D010053:Ovary / Q000166:cytology / Q000254:growth & development*; D013234:Stem Cells / Q000166:cytology*
chemical_list: D049452:Green Fluorescent Proteins; C498288:DDX4 protein, human; C090864:Ddx4 protein, mouse; D053487:DEAD-box RNA Helicases