Abstract
ObjectivesIn 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 MethodsmFGSCs 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.ResultsRT‐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.ConclusionWe evaluated five in vitro differentiation conditions for mFGSCs and successfully differentiate mFGSCs into GV‐stage oocytes using a three‐step differentiation process.
Highlights
Infertility is a reproductive health problem that affects approxi‐ mately 13%‐18% of couples in the human population.[1]
We preliminarily evaluated five different in vitro differentiation conditions for murine FGSCs (mFGSCs)
A significant fraction of cells showed differentiating oocyte‐ like morphological characteristics with enlarged diameter, suggesting mFGSCs could readily commit to differentiation in the presence of retinoic acid (RA)
Summary
Infertility is a reproductive health problem that affects approxi‐ mately 13%‐18% of couples in the human population.[1]. Researchers have reported the possibility of deriving female gametes from stem cells in vitro.[3]. After transplantation under mouse ovarian bursa, aggrega‐ tion of PGCLCs with female gonadal somatic cells generated oocytes exhibiting the capacity for fertilization that results in offspring.[9,10]. Significant breakthroughs have been made to produce female gametes derived from ESCs and iPSCs in vitro, it still remains challenging for reproductive medicine applications. Zhou et al obtained FGSCs from female rat ovaries and developed a three‐ step system to differentiate rat FGSCs into GV‐stage oocytes in vitro.[19]. No systematic comparative study of in vitro differentiation conditions for mFGSCs has been reported. Our study provides a tool to systematically identify factors and pathways involved in promoting oogenesis from FGSCs, and has exciting potential applications for reproductive and regenerative medicine
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