FACS-sorted putative oogonial stem cells from the ovary are neither DDX4-positive nor germ cells

Abstract

Whether the adult mammalian ovary contains oogonial stem cells (OSCs) is controversial. They have been isolated by a live-cell sorting method using the germ cell marker DDX4, which has previously been assumed to be cytoplasmic, not surface-bound. Furthermore their stem cell and germ cell characteristics remain disputed. Here we show that although OSC-like cells can be isolated from the ovary using an antibody to DDX4, there is no good in silico modelling to support the existence of a surface-bound DDX4. Furthermore these cells when isolated were not expressing DDX4, and did not initially possess germline identity. Despite these unremarkable beginnings, they acquired some pre-meiotic markers in culture, including DDX4, but critically never expressed oocyte-specific markers, and furthermore were not immortal but died after a few months. 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.

Enrichment of Female Germline Stem Cells from Mouse Ovaries Using the Differential Adhesion Method

“…However, whether FGSCs exist has aroused widespread controversy between supporters and opponents in recent years [10, 19, 21, 32, 33]. In 2009, since Wu et al .…”

“…Compared to somatic cells in the ovary, there are relatively few FGSCs (0.014%) in ovaries [11], and utilizing an inappropriate molecule to label putative germline stem cells, could lead to failure in isolating FGSCs [10, 19, 22, 34, 43]. Although Wu's group has used Fragilis-Based MACS to improve the efficiency of FGSC purification [44], Fragilis is also present in the oviduct, stromal cells and oocytes, confirming that this germline marker is not germline-specific [19]. Thus, the limiting factor for the study of FGSC behaviour and characteristics in vitro has been separation and purification of these cells while maintaining their self-renewal ability.…”

“…Larissa et al . reported that FACS-sorted putative oogonial stem cells from the ovary are neither Ddx4-positive cells nor germ cells [19]. Lei et al .…”

Oocyte stem cells: fact or fantasy?

“…Online transmembrane domain predictors were used on both the human and mouse Ddx4 protein, finding no evidence of a transmembrane region of human Ddx4 that could be used to isolate cells. One model predicted an extracellular domain in mice, but the overall evidence of a transmembrane Ddx4 region that could be isolated using antibodies was weak (Zarate-Garcia et al 2016). Further investigation found that Ddx4 detection may be falsely identifying cells that do not express Ddx4 (ZarateGarcia et al 2016).…”

Differentiation of Mouse Primordial Germ Cells into Functional Oocytes In Vitro

“…Although the ratio of sorted OSCs after FACS or MACS was low in these studies, OSCs proliferated with the expression of both germ cell and stem cell markers during culture, and contributed to oocytes after grafting in ovaries ex vivo . However, the use of an MVH antibody in a live-cell sorting assay to detect antigens on the cell surface is questionable because MVH is a germline-specific RNA helicase and is believed to exist in the cytoplasm 13,25,108. Several attempts to resolve this issue with the MVH antibody approach have been reported using an SSEA-1 antibody or Ddx4-cre transgenic mice 42,75.…”