The Roles Of Figla In The Derivation Of Female Germ-like Cells From Mouse Embryonic Stem Cells

Germ cells are highly specialized cell populations that are indispensable for thecontinuation and evolution of the species. An increasing number of couples suffer frominfertility and sterility because of ovarian failure or diminished ovarian reserve. The processesof both normal and abnormal oocyte development need to be interpreted. But human germcell development has been shown difficulty to study because the important early events occurafter implantation in womb. Embryonic stem cells (ESCs) are derived from the inner cellmass of the blastocyst, which could differentiate into almost all cell types in the body, andthus potentially provide unlimited cells for organ and cell replacement therapies. Previousstudies have demonstrated the germ cells, even oocyte-like cells and ovarian-like structurescan be derived from mouse ESCs. But the critical mechanisms of ESC-derived germ cellsespecially oocyte development, have not been well-documented and the efficiency was verylow. Figla (Folliculogenesis-specific basic helix-loop-helix) gene encodes a germ cell specifictranscription factor that coordinates the expression of the oocyte-specific zona pellucida (Zp)genes and is essential for folliculogenesis in mouse. Here, we first constructed a pFigla-EGFPrecombinant plasmid that expressed enhanced green fluorescent protein (EGFP) under thecontrol of Figla promoter, and generated and characterized an ESC line stably carrying thispFigla-EGFP reporter construct. Then the technique for the differentiation of female germcells from ESCs was evaluated. The effect of Figla on the promotion of the germ cellderivation from ESCs, and the regulation of some female germ cell specific genes expressionwas detected.1. Selection of ESCs transfected with pFigla-EGFPWe analyzed the promoter region for the Figla gene using the Promoter On-line AnalysisDatabase, constructed a pFigla-EGFP recombinant plasmid that expressed EGFP under thecontrol of Figla promoter, and generated and characterized an ESC line stably carrying thispFigla-EGFP reporter construct. The ESC line maintained pluripotent characterization anddifferentiation potentiality.2. The differentiation of female germ cells from ESCs induced by different methodsWe induced mouse ES cells to differentiate into oocyte-like cells by culturing adherentEBs in several different medium, including retinoic acid (RA), follicular fluid and FSH. Wealso cocultured EBs with ovarian cells, or transplanted ESCs under the kidney capsule with ovarian cells to allow the differentiation. The extent of differentiation was evaluated throughmorphology and the expression of certain specific germ cell markers. As a result, germ cellprecursor cells were first differentiated from ESCs, and subsequently EBs developed intoovarian-like structures that contained oocyte-like cells showed expression of germ cellspecific markers on the differentiation condition of RA, follicular fluid and coculture. Someof the kidney capsule transplanted ESCs differentiated into large cells, forming folliclestructures. A population of differentiated ESCs expressed GFP, and these cells simultaneouslyshowed the expression of germ cell and oocyte markers indicating that this method could beused for the purification and selection of female germ cells.3. Overexpression of Figla induces differentiation of ESCs toward female germ cellsWe cloned the Figla gene and constructed its eukaryotic vector, which was namedpDsRed1-N1-Figla and transfected into ESCs. The results showed that cells expressed redfluorescent protein (RFP), and germ cell specific genes were up-regulated. Our study showedthat overexpression of Figla can promote the differentiation of ESCs toward female germ cells,and Figla can regulate the expression of some female germ cell specific genes.