视黄酸信号通路在青鳉精原干细胞体外增殖与分化中的作用

Role of retinoic acid signaling in the proliferation and differentiation of medaka(Oryzias latipes)spermatogonial stem cells in vitro

为探究视黄酸(RA)在鱼类精原干细胞(SSC)增殖与分化中的作用,实验首先以三色荧光报告载体pGRY[延伸因子1α启动子驱动组蛋白H2B-绿色荧光融合蛋白、减数分裂联会复合体蛋白3(Scp3)启动子驱动嘌呤霉素红色荧光融合蛋白、精蛋白启动子驱动黄色荧光蛋白]转染青鳉精原干细胞系SG3,获得稳转细胞SG3-pGRY,然后分别在2D与3D培养条件下检测RA信号对其增殖与分化的影响。结果显示,稳转细胞SG3-pGRY的红色荧光可监测细胞内源性Scp3的表达,且细胞仍保持干性及分化潜能,表明其可用于监测细胞分化状态。在2D培养条件下,即在细胞培养板中待细胞生长密度约90%就进行传代培养,RA可显著抑制细胞增殖,其受体α、β、γ泛抑制剂BMS493可促进细胞增殖。第48小时,RA处理可下调细胞多能性相关基因pou5f3、klf4表达,上调减数分裂相关基因dazl表达,但对其他减数分裂相关基因如scp3表达无明显影响。第8天,处理组及对照组均未能观察到红色荧光,这与已有报道RA处理体外培养小鼠SSC可显著促进Scp3表达,并进入减数分裂I期偶线期的研究结果明显不同。在3D培养条件下,即用96孔球形低吸附微量培养板进行培养,48 h后细胞成球状体聚集生长,RA、BMS493处理组、对照组在48 h后均观察到明显红色荧光,减数分裂相关基因表达与2D相比显著上调。第8与第34天,RA处理组减数分裂相关基因表达均显著高于对照组,而BMS493处理组低于对照组。研究表明,RA信号可抑制SG3增殖,促进其分化,但不是诱导细胞发生减数分裂的关键分子。本研究不仅为鱼类SSC分化相关研究提供了良好研究模型,而且促进了对于RA信号在鱼类SSC增殖与分化中作用的深入认识。

Studies have shown in mammals that retinoic acid(RA)is a key molecule mediating the initiation of meiosis,and can directly induce meiosis in cultured spermatogonial stem cells(SSCs)and express meiosis spe-cific molecule Scp3.However,although studies have shown that RA plays an important role in testis differenti-ation and development and meiosis initiation in fish,its role in SSC proliferation and differentiation and whether it can directly mediate SSC meiosis remain unclear.In this study,the three-fluorescence reported plasmid pGRY,which contains three promoters,i.e.,elongation factor 1α(ef1α)promoter driven histone H2B-green fluorescent protein(H2B-GFP),scp3 promoter driven puromycin-red fluorescent protein(puro-RFP),and protamine promoter driven yellow fluorescent protein(YFP),was used to transfect SG3 to obtain stable cell line SG3-pGRY.Then the effect of RA signal on the proliferation and differentiation of SG3-pGRY was investigated.The red fluorescent of SG3-pGRY could indeed reflect the expression of endogenous Scp3.Moreover,the SG3-pGRY retained stem property such as strong alkaline phosphatase activity and differentiation potential,indicating that it can be used to monitor the state of cell differentiation.With the condition of 2D culture,that is to say,the cells are sub-cultured when the cell growth density is about 90%in the cell culture plate,RA could inhibit cell proliferation,while Rar(α,βandγ)pan-antagonist BMS493 could promote cell proliferation.Furthermore,the results of real-time PCR showed that RA could significantly down-regulate the expression of pluripotency related genes pou5f3 and klf4 and had no significant effect on the expression of differentiated related genes including ckit,scp3,spo11,rec8a,rec8b except dazl.With the condition of 3D,obvious red fluorescence was observed in RA,BMS493 and control group after 48 h incubation.Compared with 2D conditions,the expressions of differentiation related genes were up-regulated compared with those in 2D culture for 48 h.In addition,the expressions of differentiation related genes in RA group were higher than the control group,whilst the BMS493 group was lower than control group.Taken together,RA signal could inhibit the proliferation and promote the differentiation of SSCs,but it is not essential to induce cell meiosis.This study not only provides a good research model for the study of fish SSC differentiation,but also promotes our in-depth understanding of the role of RA signal in fish SSC proliferation and differentiation.