205 Preliminary characterization of ovarian stem cells from bovine ovaries

Ovarian stem cells (OSCs) have been reportedly isolated from ovaries of rodents, pigs, humans, and cattle by targeting the germ cell marker protein DDX4. Although the role of OSCs in female reproduction is unknown, the ability to culture OSCs and differentiate oocytes in vitro could benefit the cattle industry and the study of oogenesis. The aim of this study was to describe isolation and preliminary characterisation of putative bovine OSCs. Slaughterhouse-derived ovaries from adult cows were processed by mechanical and enzymatic dissociation into a single cell suspension followed by immunostaining. Cells were incubated in blocking solution followed by 10 µg mL−1 rabbit anti-human polyclonal DDX4 antibody (#13840; Abcam) for 15 min and 2 µg mL−1 goat anti-rabbit IgG labelled with Alexa Fluor 647 for 15 min in the dark. Next, cells were resuspended in Hanks’ balanced salt solution with 1% bovine serum albumin/25 mM HEPES, filtered through a 30-µm strainer and subjected to fluorescence-activated cell sorting. Controls used to establish gates were unstained cells and cells incubated with secondary antibody only. 4’,6-Diamidino-2-phenylindole (DAPI) exclusion was used as a viability test. Putative OSCs were placed in culture in OSC medium (MEMα Glutamax containing 10% fetal bovine serum, 1 mM sodium pyruvate, 1× nonessential amino acids, 103 units of leukemia inhibitory factor, 10 µg mL−1 glial cell-derived neurotrophic factor, 10 µg mL−1 basic fibroblast growth factor, 1 µg mL−1 epidermal growth factor, 1× N2-max, penicillin/streptomycin) for expansion and characterisation by gene expression using reverse transcription-PCR and protein expression using immunolocalization and confocal microscopy. To ensure specificity against bovine DDX4, the same antibody used for cell sorting was used to label oocytes within ovarian follicles in histological sections. Two cell lines were obtained and expanded in vitro. Gene expression was performed in putative OSCs at passages 1 to 3; cumulus-oocyte complexes (COCs) were used as positive controls and adult skin fibroblasts as negative controls, and ACTB was used as an endogenous control. Both putative OSC lines and COCs expressed the germ cell markers DAZL and C-KIT, and COCs also expressed BMP15. Only ACTB was detected in fibroblasts. Immunolocalization was performed in putative OSCs at passage 4, with oocytes and fibroblasts used as positive and negative controls. Additional controls were cells exposed to secondary antibody only. Both putative OSC lines and oocytes expressed DAZL and DDX4 and no marker was detected in fibroblasts. Next, OSC line #2 was transfected with a retroviral vector using the EF1α promoter for green fluorescent protein (GFP) expression. This is a critical step to ensure the success of experiments requiring cell tracking. Transfected cells were expanded and sorted to establish a pure population of GFP+ OSCs. To verify replication deficiency of the viral particles, supernatant from GFP+ OSCs was collected 1 passage after transfection and applied to GFP- OSCs. No GFP+ cells were observed after 24, 48, or 72 h. These preliminary results confirm the presence of putative OSCs in the ovaries of cows of reproductive age. If these cells are capable of in vitro differentiation, they could provide a powerful tool to study oogenesis and further develop assisted reproductive technologies.