Comparison of different in vitro differentiation conditions for murine female germline stem cells

Abstract

Objectives: In vitro differentiation of oocytes from female germline stem cells (FGSCs) has exciting potential applications for reproductive medicine. Some researchers have attempted to reveal the in vitro differentiation capacity of FGSCs. However, no systematic comparative study of in vitro differentiation conditions has been performed for murine FGSCs (mFGSCs). Materials and Methods: mFGSCs line was cultured under five different conditions for in vitro differentiation. RT-PCR was performed to detect the expression of Oct4, Fragilis, Blimp1, Mvh, Scp3 and Zp3. Immunofluorescence was carried out to test the expression of Mvh, Fragilis and Zp3. Two-photon laser-scanning microscope was used to analyze nucleus-plasma ratio, and the proportion of chromatin of GV oocytes differentiated from mFGSCs in vitro (IVD-GVO), GV oocytes from in vivo (GVO) and mFGSCs. Results: RT-PCR and immunofluorescence showed that mFGSC line expressed germ cell-specific markers, but not a meiosis-specific marker. By evaluating five different in vitro differentiation conditions, condition 5, which included a hanging drop procedure and co-culture of mFGSCs with granulosa cells, was shown to be optimal. mFGSCs could be successfully differentiated into germinal vesicle (GV) -stage oocytes under this condition. 3D observation revealed that both the nucleus-plasma ratio and proportion of chromatin were not significantly different between IVD-GVO and GVO. Conclusion: We evaluated five in vitro differentiation conditions for mFGSCs and successfully differentiate mFGSCs into GV-stage oocytes using a three-step differentiation process.