Figla基因过表达促进小鼠胚胎干细胞向雌性生殖细胞分化

Figla Overexpression Induces Differentiation of Mouse Embryonic Stem Cells toward Female Germ Cells

生殖系a因子(Figla)是最早表达的生殖细胞特异性转录因子之一,对卵泡的发育、Zp基因的表达和透明带的形成具有调节作用.Figla基因异常会引起卵巢早衰的发生.本研究通过PCR自小鼠基因组中扩增出Figla基因,将其克隆到真核报告载体pDsRed1-N1,构建了携带609 bp的Figla重组载体pDsRed1-N1-Figla.用该载体转染小鼠胚胎干细胞(mESCs)系J1、小鼠成纤维细胞系NIH-3T3、小鼠畸胎瘤细胞P19和小鼠精原细胞系GC1,在荧光显微镜下观察红色荧光蛋白(RFP)在细胞中的表达,同时检测转染细胞中Figla基因及其它生殖细胞特异性基因的表达.结果显示,转染2 d,mESCs内Figla总表达量明显增加,且内源性表达量亦有所提高,即转入的外源性Figla基因可以促进内源性Figla的启动和表达.免疫荧光染色显示,表达RFP的细胞同时表达生殖特异性基因Vasa,减数分裂特异性基因Stra8、Scp3及卵母细胞标志基因Zp3.通过QRT-PCR检测发现,在转染3 d的细胞中,Vasa、Scp3和Zp1的表达较对照组均有明显上调,而Oct4和Stra8的表达量下降.研究表明,Figla基因对生殖特异性基因的表达具有调控作用,可以激活雌性生殖基因表达,为更清楚地了解Figla基因在生殖细胞生长发育过程中的调控机制,以及发现该基因在生殖细胞中的新功能奠定了基础.

Figla（factor in germline alpha）,a basic helix-loop-helix（bHLH） transcription factor,plays a crucial role in the formation of primordial follicles.Figla regulates the expression of multiple oocyte-specific genes,such as the zona pellucida（Zp） proteins Zp1,Zp2,Zp3,and the formation of the Zona pellucida.Female mice lacking Figla are unable to form primordial follicles resulting in massive depletion of oocytes and sterility.In human,premature ovarian failure（POF） harbors mutations in Figla.We cloned the Figla gene and constructed its eukaryotic expression vector,named pDsRed1-N1-Figla and transfected into ES cells,3T3,P19 and GC1.The expression of the recombinant vector can be observed by red fluorescent protein（RFP） using the fluorescent microscope.The results showed that the eukaryotic expression vector pDsRed1-N1-Figla had been successfully transfected into cells and both exogeneous and endogenous of Figla expression up-regulated.Cells expressed RFP could be distinguished from the other cells by their large size,round shape and blebbing,morphologically differentiated into oocyte-like cells.Immunofluorescence staining showed that the RFP positive cells expressed germ cell specific gene Vasa,meiosis specific genes Stra8,Scp3 and oocyte marker Zp3.QRT-PCR analysis showed that Vasa,Scp3 and Zp1 were highly expressed in pDsRed1-N1-Figla transfected cells,while Oct4 and Stra8 were down-regulated compared to the control.The results showed that Figla regulated the expression of some germ cell specific genes,which may offer an approach for further research on regulatory mechanism of Figla in female germline and follicle development.