Understanding the meiotic competence of oocytes derived from oogonial stem cells

Abstract

This thesis investigates whether Oogonial Stem Cells (OSCs) exist and can be isolated from the adult mouse ovary. It also examines their ability to differentiate into oocyte-like cells in vitro. Transmembrane domains in the DEAD-box polypeptide 4 (DDX4) – the germline marker used in this antibody-based isolation – are examined using in silico protein modelling. The specificity of this antibody to DDX4 is tested in male and female germline cells. It is shown that the DDX4 antibody may have some specificity for DDX4, but the existence of a surface-bound DDX4 is unlikely. OSC-like cells can be isolated from the ovary using the DDX4 antibody by Fluorescence-Activated Cell Sorting (FACS). However, gene expression analysis and protein immunofluorescence show that these cells do not initially express DDX4 or possess germline identity. Despite this, they acquire some pre-meiotic and oocyte-specific markers in culture, including DDX4. Critically, the cells never express meiosis-specific markers even in the presence of meiotic enhancers BMP4 and retinoic acid. It is unlikely that these ovarian cells are being sorted by means of a cell surface DDX4 expression, because another antibody to a larger DDX4 epitope fails to detect DDX4 in isolated cells. These findings highlight that freshly isolated OSCs are not germ stem cells, and are not being isolated by their DDX4 expression, but instead may reflect an artefact of the antibody used or procedure adopted. Altogether this work offers support to the established dogma that the adult ovary is populated at birth by a fixed number of oocytes, and that adult de novo production is a rare or insignificant event.

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ORCID for Keith Jones: orcid.org/0000-0002-0294-0851

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