Investigating the oogenic potential of bovine oogonial stem cells

Abstract

A fixed population of oocytes within primordial follicles, established prior to or just after birth has been firmly believed to support the postnatal mammalian ovary throughout an individual’s reproductive lifespan. However, the identification and isolation of cells from adult mammalian ovaries characterised by the expression of both germ and stem cell markers, suggest the presence of mitotically active cells, termed oogonial stem cells (OSCs) that may have the potential to produce new oocytes in the postnatal mammalian ovary. Putative OSCs have been isolated from adult tissues of several mammalian species, including rodents and humans. Upon reintroduction to the ovarian niche, human and rodent OSCs have generated new oocyte like structures which, at least in rodents, have generated functional oocytes capable of fertilisation and subsequent embryonic development to produce healthy offspring. We hypothesised that OSCs could be isolated from adult bovine ovaries and upon establishment within the appropriate ovarian niche could initiate successful oogenesis. To investigate this hypothesis, we have utilised fluorescently activated cell sorting (FACS) to isolate putative bovine OSCs (bOSCs) and an ovarian aggregate model, in vitro and in vivo to explore the oogenic potential of these cells. Putative bOSCs were successfully isolated by FACS based on the cell surface expression of germ cell marker DDX4 and established in culture. Pluripotency (LIN28 and OCT4) and germ (IFITM3, PRDM1, C-KIT and DAZL) cell associated genes were expressed in putative bOSCs established in culture. However, DDX4 transcripts were not consistently observed throughout bOSC culture. Aggregation of putative bOSCs with neonatal murine ovarian somatic cells to form chimeric ovarian aggregates, cultured in a hanging drop model for 7 days maintained germ cell phenotype, marked by DAZL expression. A subpopulation of putative bOSCs showed a spherical morphology, an increase in cell size and an association with neighbouring cells. Xenotransplantation of chimeric ovarian aggregates to the kidney capsule of immune deficient mice for 21 days generated multi-laminar follicles and structures with morphological similarities to primordial follicles (termed pre-primordial follicle-like structures). RNA Scope was unsuccessful in determining the origin of oocytes within chimeric ovarian aggregates. However, oocytes from pre-antral follicles in chimeric ovarian aggregates (n=6; 60.9± 3.6 μm, mean ± SEM) were significantly (P<0.0001) larger than murine oocytes (n=38; 34.5± 1 μm, mean ± SEM) aggregated with murine ovarian somatic cells as positive controls, suggesting that these oocytes are undergoing different growth dynamics. This work has shown that putative bOSCs characterised by the expression of pluripotency and germ cell associated genes are present within adult bovine ovarian tissue and can be isolated using FACS and established in culture. These data also suggest that putative bOSCs may have the potential to undergo oogenesis and illustrate the potential use of these cells as a tool to investigate germ cell differentiation.