Establishment of female germline stem cells derived from bovine ovary and differentiation to oocyte-like cells

Abstract

Female germline stem cells (FGSCs) have been successfully established and differentiated into oocytes in small animals. However, whether FGSCs can be established and differentiated to oocyte-like cells (OLCs) in large animals like bovine is still unclear. Therefore, the aim of this research is to establish FGSCs from bovine ovaries and differentiate them to OLCs. In this experiment, cortical tissues were cut from the ovarian surface epithelium of bovine ovaries to isolate FGSCs. The isolated FGSCs were characterized with germ cell marker DAZL and continued to exam the differentiation potential to oocyte-like cells by co-culture with granulosa cells isolated from pre-antral bovine follicles. The growth factors such as estradiol, androgen and follicle-stimulating hormone (FSH) also being tested and applied for culture medium of FGSCs via in vitro growth of small oocytes. Furthermore, primordial oocytes also be collected from the bovine ovaries as a control group to compare with OLCs by immunostaining with germ cell marker VASA and oocyte marker GDF9. As a result, the FGSCs isolated from 1-week culture expressed DAZL marker and the enlarged cells (up to 20-30μm in diameter) picked up from 7-day co-culture dish expressed both VASA and GDF9 in their cytoplasm. In addition, the co-culture with granulosa cells also shows positive result in promoting the differentiation to OLCs comparing with mouse embryonic fetus (MEF) feeder layer. In conclusion, this research have successfully isolated and characterized bovine FGSCs but the differentiation to OLCs still need more improvement. We suggested that the co-culture with granulosa cells can enhance the FGSCs differentiation to OLCs. Keyword: FGSCs, OLCs, maintenance, differentiation, oogenesis, granulosa cells, immunostaining.