Adult human and mouse ovaries lack DDX4-expressing functional oogonial stem cells

The generally accepted viewpoint for more than 50 years has been that the number of oocytes is fixed in fetal or neonatal ovaries, and therefore, oocytes cannot renew themselves in postnatal or adult life. Over the past decade, however, the traditional viewpoint has been challenged by a number of investigators who have presented evidence that postnatal follicular renewal occurs in mammals, and that mitotically active oogonial stem cells (OSCs) exist in postnatal mouse ovaries. Health, Inc. All rights reserved. 30 Obstetrical and Gynecological Survey This letter to the editor presents experimental evidence that disputes the existence of mitotically active OSC in postnatal mouse ovaries. The results presented here are the summary of research conducted independently in 4 laboratories. A previous study (White et al.Nat Med. 2012;18:413–421) reported that OSCs could be purified from adult human and mouse ovaries by use of DEAD box polypeptide 4 (DDX4) antibody–based fluorescence-activated cell sorting (FACS), and that after in vitro manipulation, these isolated OSCs could form oocytes. Based on the well-established cytoplasmic location of DDX4, the use of this protein as a cell surface marker is controversial. Using the same DDX4 antibody–based FACS approach as in the White et al study, the investigators isolated a population of cells from human ovarian cortical tissue biopsied from 16 fertile reproductive-age women who had had at least 1 previous live birth. No DDX4 messenger RNA (mRNA) expression was detected using quantitative polymerase chain reaction in these cells or by a more sensitive single-cell mRNA sequencing analysis that could detect low expression of DDX4. In additional experiments, the sorted human ovarian cells were cultured as described in the previous study. Although no DDX4 expression was detected in the cultured DDX4-positive cells (cultured-POS) or cultured DDX4-negative cells (cultured-NEG) by immunofluorescence staining, the cultured-POS cells and cultured-NEG cells both bound tightly to the DDX4-specific antibody in FACS and became DDX4-positive after culture. The previous study had reported that oocytes enclosed in follicles regenerated 1 week after the DDX4-positive human OSCs were injected into human ovarian cortical tissues that were subsequently xenografted into female severe combined immunodeficient mice. The investigators repeated this experiment and labeled the cultured-POS cells with stable enhanced green fluorescent protein (EGFP) expression. After culturing and expanding this cell population, EGFP-expressing cultured-POS cells were injected into human ovarian cortical tissue biopsies, and these cortical tissues were then xenografted into female severe combined immunodeficient mice for further growth. Grafts were analyzed 1 week, 2 weeks, and 4 weeks after transplantation of the EGFP–cultured-POS cells into the human cortical tissues. The results of this experiment showed that EGFP-positive cells could be observed in the vicinity of the injection sites, but the absence of any EGFP-positive oocytes demonstrated that the DDX4-positive human cells obtained with the DDX4 antibody are not functional stem cells and cannot regenerate oocytes. To confirm these findings that the DDX4-specific antibody–based FACS does not select for a specific cell population expressing DDX4, the same FACS was performed with mouse cells from several organs (including adult liver, spleen, and kidney) that do not express DDX4. DDX4-positive cell populations were obtained from cells of these organs, which provide additional evidence that use of the DDX4-specific antibody using the FACS protocol (critical in purifying the reported OSCs) does not select for DDX4-expressing cells. These findings provide evidence that supports the traditional view that no postnatal follicular renewal occurs in mammals, and no mitotically active DDX4-expressing female germline progenitors exist in postnatal mouse ovaries.