性激素促进鸡胚胎干细胞向雄性生殖细胞分化  

Study on Differentiation of Chicken Embryonic Stem Cells to Male Germ Cells by Sex Hormone

作　　者：张文慧[1] 蒋一秀 王颖洁[1] 左其生[1] 李东[1] 连超[1] 汤贝贝[1] 张亚妮[1] 李碧春[1] 

机构地区：[1]扬州大学动物科学与技术学院,江苏省动物遗传繁育与分子设计重点实验室,江苏扬州225009

出　　处：《中国家禽》2016年第8期10-16,共7页China Poultry

基　　金：国家自然科学基金项目(31272429、31472087、31301959);江苏省高校优势学科建设工程资助项目

摘　　要：试验分别探讨了睾酮和FSH诱导鸡胚胎干细胞(Embryonic stem cells,ESCs)向雄性生殖细胞分化的作用效果。首先筛选睾酮和卵泡刺激素(FSH)的适宜诱导浓度,在RA和支持细胞的诱导基础上分别添加适宜浓度的睾酮和FSH对传至2代的ESCs进行诱导,通过形态学观察、实时荧光定量PCR及免疫细胞化学对诱导结果进行检测。结果显示:睾酮和FSH的适宜诱导浓度分别为15 ng/m L和25 ng/m L,单独使用睾酮和FSH诱导均未出现类SSCs样细胞;二者联合支持细胞和RA诱导,诱导2 d出现类胚体,随后类胚体体积逐渐变大,诱导12 d类胚体解体出现类SSC样细胞;实时荧光定量PCR结果显示,睾酮和FSH分别与支持细胞和RA共同诱导组中,Stra8表达量持续上调,integrinβ1、Dazl表达量显著上调,ESCs标记基因Nanog、Sox2表达量均呈持续下降趋势;睾酮的多因素诱导组中,Dazl的表达量明显高于RA和支持细胞诱导组;而FSH的多因素诱导组中Stra8的表达量相对于RA和支持细胞诱导组也显著升高。结果表明睾酮和FSH单独诱导不能引起ESCs向雄性生殖细胞分化,但可以促进支持细胞和RA诱导ESCs向雄性生殖细胞分化的作用效果,该结果为进一步研究雄性生殖细胞的形成和调控机制提供理论基础。This study investigated the effects of testosterone and FSH on differentiation of embryonic stem ceils （ESCs） to male germ cells respectively. The suitable concentrations of testosterone and FSH were screened firstly by experiments, then added them into the medium with RA and sertoli cells to induce 2 passaged maleness ESCs respectively. The induced effects were detected by morphology, quantitative polymerase chain reaction （qPCR）and immunocytochemistry. The suitable concentrations of testosterone and FSH were 15 ng/mL and 25 ng/mL respectively. To induce the ESCswith only testosterone or FSH. The germ cell-like cells could not appeared, embryoid bodies （EBs）could be observed in 2 d in the groups of testosterone or FSH combined with RA and sertoli cells, then EBs were gradually increased in both size and number,then EBs broke and germ cell-like cells could be observed in 12 d. The result of qPCR showed that the expression levels of germ cell-specific genes StraS,integrinl31 ,Dazl in the two groups all showed a significant up regulation, while the expression level of the ESCs marker genes Nanog, Sox2 decreased significantly. The expression of Dazl in testosterone with multiple factors inducing group was significantly higher than that in RA and support cells induced group;and the expression of Stra8 had the similar trend in FSH with multiple factors inducing group as Dazl. Testosterone and FSH had effects on differentiation of ESCs to male germ cells. The expressions of the corresponding genes of the germ cells were higher than them in the group with RA and sertoli cells. The resuhs provide theoretical foundation for studying cytogenesis and regulatory mechanism of male germ cells.

关 键 词：鸡胚胎干细胞 雄性生殖细胞 分化 睾酮 卵泡刺激素 

分 类 号：S813.3[农业科学—畜牧学]