Figla在尼罗罗非鱼卵巢分化和维持过程中的作用

Functional studies of Figla in the ovarian differentiation and maintenance in Nile tilapia(Orechromis niloticus)

为了深入研究Figla在卵巢分化早期的功能,实验通过转录激活因子样效应物核酸酶(transcription activator-like effector nucleases,TALEN)介导的基因组靶向修饰技术在雌性尼罗罗非鱼体内成功敲除了Figla基因。结果显示,TALEN能高效地对Figla基因进行靶向性编辑,并通过测序筛选到不同碱基数目(4,8,20等)缺失的基因型个体。组织学观察发现Figla基因敲除阳性XX个体在3月龄时出现卵巢发育异常,表现为卵母细胞和滤泡细胞大量或完全缺失,并伴随体细胞的大量增生。免疫组化结果显示,在Figla基因敲除阳性个体的卵巢中检测到雄激素合成酶(Cyp11b2)的表达,但未检测到雌激素合成酶(Cyp19a1a)的表达。血清11-KT水平升高,而E2水平随之降低。研究认为Figla在硬骨鱼类卵巢的分化和维持过程中发挥至关重要的作用。

Figla（factor in the germ line, alpha）, encoding a germ cell-specific transcription factor, is a central regulator of oocyte-specific genes. In order to explore the potential role of Figla in fish gonadal differentiation,tilapia Figla gene was successfully targeted by TALENs（transcription activator-like effector nucleases） in XX individual and confirmed by genomic PCR and subsequent enzyme cleavage and sequencing. Consequently, the morphological and molecular changes in XX gonads were observed by H.E, IHC. Meanwhile, the alterations of E2 and 11-KT were also determined. From screening of different mutant genotypes with bases deletions（4, 8, 20,etc.）, it was revealed that the efficiency of Figla gene targeting by TALEN was about 56.76%. Furthermore,deficiency of Figla gene in XX fish led to development of abnormal ovaries with severe degeneration or complete loss of oocytes and ectopic proliferations of somatic cells. Amazingly, IHC in Figla-deficient XX gonads revealed that male specific marker gene（Cyp11b2） for 11-KT production in leydig cells was expressed in the cells surrounding oocytes, however, Cyp19a1 a was barely detectable which is strongly expressed in follicular cells of normal ovary. Notably, Figla deficiency led to dramatic decrease of E2 serum level along with augmentation in serum 11-KT level. Taken together, our data indicated that Figla might play an essential role in the ovarian differentiation and maintenance in tilapia fish.