Implications and Current Limitations of Oogenesis from Female Germline or Oogonial Stem Cells in Adult Mammalian Ovaries

Abstract

A now large body of evidence supports the existence of mitotically active germ cells in postnatal ovaries of diverse mammalian species, including humans. This opens the possibility that adult stem cells naturally committed to a germline fate could be leveraged for the production of female gametes outside of the body. The functional properties of these cells, referred to as female germline or oogonial stem cells (OSCs), in ovaries of women have recently been tested in various ways, including a very recent investigation of the differentiation capacity of human OSCs at a single cell level. The exciting insights gained from these experiments, coupled with other data derived from intraovarian transplantation and genetic tracing analyses in animal models that have established the capacity of OSCs to generate healthy eggs, embryos and offspring, should drive constructive discussions in this relatively new field to further exploring the value of these cells to the study, and potential management, of human female fertility. Here, we provide a brief history of the discovery and characterization of OSCs in mammals, as well as of the in-vivo significance of postnatal oogenesis to adult ovarian function. We then highlight several key observations made recently on the biology of OSCs, and integrate this information into a broader discussion of the potential value and limitations of these adult stem cells to achieving a greater understanding of human female gametogenesis in vivo and in vitro.

Keywords: fertility; germ cell; germline stem cell; meiosis; oocyte; oogenesis; oogonial stem cell; ovary.

Figure 1

Oocytes formed during adult life in mice contribute directly to offspring production. Schematic representation of an inducible genetic lineage-tracing model to ‘mark’ new oocytes formed during the doxycycline (dox) induction phase, which specifically activates a triple-transgenic Cre-loxP–based reporter system tied to stimulated by retinoic acid gene 8 (Stra8) expression in pre-meiotic germ cells (viz. OSCs) committing to meiosis followed by de novo oogenesis. Portions of this figure were adapted with permission from Wang et al. [84].

Figure 2

In vitro oogenesis from human OSCs. (A) Time lapse images of human OSCs in culture, with a typical OSC (blue arrow) followed as it undergoes progressive differentiation into an IVD oocyte (oocyte-like cell). (B) Numbers of IVD oocytes formed in human OSC cultures over time post-passage. (C) Expression analysis of germ cell (DDX4) and oocyte (KIT oncogene or KIT; Y-box protein 2 or YBX2, also referred to as MSY2 or CONTRIN; LIM homeobox protein 8 or LHX8) marker genes, as well as of β-actin expression as a loading control, in IVD oocytes collected from human OSC cultures (–RT, PCR analysis performed on the RNA template without reverse transcription, as a control to rule out genomic DNA amplification). (D) Representative images of human IVD oocytes by light microscopy (two left panels; scale bar, 50-µm), and by immunofluorescence microscopy for the presence of DDX4, KIT, YBX2 and LHX8 proteins. Portions of this figure were adapted with permission from White et al. [73].

Figure 3

Mouse VSEL stem cells do not express externalized Ddx4. (A) To identify VSEL stem cells by FACS, size gates were initially set for 2–10 µm using calibrated beads. (B, C) Following dead cell exclusion, bone marrow-derived VSEL stem cells were identified in the 2–10 µm size gate as Lin⁻/Sca1⁺/CD45⁻ events, first by gating Lin⁻/Sca1⁺ cells followed by exclusion of CD45⁺ cells from this subpopulation (for additional details, see [129]). (D) Further analysis of the VSEL stem cell population shown in panel C using C-terminal-directed Ddx4 antibodies demonstrates that VSEL stem cells do not express externalized Ddx4 (ecDdx4⁻). (E) As a positive control for the VSEL stem cell analysis shown in panel D, parallel sorting of dispersed ovaries identifies viable ecDdx4⁺ cells, which represent OSCs (for additional details, see [68,73,112]). D.C. Woods and J.L. Tilly, unpublished data.