Abstract
Putative oogonial stem cells (OSCs) have been isolated by fluorescence-activated cell sorting (FACS) from adult human ovarian tissue using an antibody against DEAD-box helicase 4 (DDX4). DDX4 has been reported to be germ cell specific within the gonads and localised intracellularly. White et al. (2012) hypothesised that the C-terminus of DDX4 is localised on the surface of putative OSCs but is internalised during the process of oogenesis. This hypothesis is controversial since it is assumed that RNA helicases function intracellularly with no extracellular expression. To determine whether the C-terminus of DDX4 could be expressed on the cell surface, we generated a novel expression construct to express full-length DDX4 as a DsRed2 fusion protein with unique C- and N-terminal epitope tags. DDX4 and the C-terminal myc tag were detected at the cell surface by immunocytochemistry and FACS of non-permeabilised human embryonic kidney HEK 293T cells transfected with the DDX4 construct. DDX4 mRNA expression was detected in the DDX4-positive sorted cells by RT-PCR. This study clearly demonstrates that the C-terminus of DDX4 can be expressed on the cell surface despite its lack of a conventional membrane-targeting or secretory sequence. These results validate the use of antibody-based FACS to isolate DDX4-positive putative OSCs.
Highlights
The field of reproductive biology is divided over the possibility of neo-oogenesis in the adult mammalian ovary [1]
DEAD-box helicase 4 (DDX4) Is Expressed in Small Ovarian Cells, in Addition to Human Oocytes
Discrete positive DDX4 staining was detected in small human ovarian cells (3–15 μm diameter) not associated with follicular structures; this was observed with both antibodies (Figure 1Bi–iv)
Summary
The field of reproductive biology is divided over the possibility of neo-oogenesis in the adult mammalian ovary [1]. In 2004, Tilly and colleagues challenged this [3], and subsequent data have provided evidence for the existence of a population of putative oogonial stem cells (OSCs) in mice [4,5,6,7,8,9,10,11,12,13], rats [14], cattle [15] and humans [6,15,16,17,18,19,20,21,22,23,24] These have the potential to form functional oocytes both in vitro [4–8,14,16,17,19–. This was used in conjunction with magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS) to isolate
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