Whether the adult mammalian ovary contains oogonial stem cells (OSCs) is controversial. They have been isolated by fluorescence-activated cell sorting (FACS) using the germline marker DEAD box polypeptide 4 (DDX4), previously assumed to be cytoplasmic, not surface-bound. Furthermore their stem cell and germ cell characteristics remain disputed. We applied a validated protocol to isolate mouse putative OSCs from whole ovarian cell suspensions, then assessed their in vitro germline and meiotic progression.
FACS-positive cells were indeed isolated using a DDX4 antibody. By immunofluorescence, they stained positive on their cell surface, but did not express measurable Ddx4 mRNA by PCR. Similarly, adult mouse oviduct showed high levels of immunofluorescence staining for DDX4, but no associated gene expression. A second independent antibody to a larger part of DDX4 was used, and both cell types stained negative. We conclude that the DDX4 antibody used for FACS sorting is not binding to a membrane-bound DDX4, but instead isolates cells through unrelated protein affinity.
The FACS-sorted cells were further interrogated for gene and protein detection of germline and oocyte markers (Prdm1, Dppa3, Ifitm3, Ddx4, Dazl, Pou5f1, Stra8, Nobox, Zp3). Despite them initially not possessing germline identity, they acquired some pre-meiotic markers in culture (Ddx4, Pou5f1), but critically never expressed markers for meiosis or oogenesis. Morphologically they never produced large rounded oocyte-like cells. Furthermore, the cells were not immortal but died within three months post-sorting.
Our results suggest that freshly isolated OSCs are not germ stem cells, and are not being isolated by their DDX4 expression. However it may be that culture induces some pre-meiotic markers. In summary the present study offers weight to the dogma that the adult ovary is populated by a fixed number of oocytes, and that adult de novo production is a rare or insignificant event.
11 Jul 2016 - 11 Jul 2016