Duplication and subfunctionalisation of the general transcription factor IIIA (gtf3a) gene in teleost genomes, with ovarian specific transcription of gtf3ab

Fish oogenesis is characterised by a massive growth of oocytes each reproductive season. This growth requires the stockpiling of certain molecules, such as ribosomal RNAs to assist the rapid ribosomal assembly and protein synthesis required to allow developmental processes in the newly formed embryo. Massive 5S rRNA expression in oocytes, facilitated by transcription factor 3A (Gtf3a), serves as marker of intersex condition in fish exposed to xenoestrogens. Our present work on Gtf3a gene evolution has been analysed in silico in teleost genomes and functionally in the case of the zebrafish Danio rerio. Synteny-analysis of fish genomes has allowed the identification of two gtf3a paralog genes, probably emerged from the teleost specific genome duplication event. Functional analyses demonstrated that gtf3ab has evolved as a gene specially transcribed in oocytes as observed in Danio rerio, and also in Oreochromis niloticus. Instead, gtf3aa was observed to be ubiquitously expressed. In addition, in zebrafish embryos gtf3aa transcription began with the activation of the zygotic genome (~8 hpf), while gtf3ab transcription began only at the onset of oogenesis. Under exposure to 100 ng/L 17β-estradiol, fully feminised 61 dpf zebrafish showed transcription of ovarian gtf3ab, while masculinised (100 ng/L 17α-methyltestosterone treated) zebrafish only transcribed gtf3aa. Sex related transcription of gtf3ab coincided with that of cyp19a1a being opposite to that of amh and dmrt1. Such sex dimorphic pattern of gtf3ab transcription was not observed earlier in larvae that had not yet shown any signs of gonad formation after 26 days of oestradiol exposure. Thus, gtf3ab transcription is a consequence of oocyte differentiation and not a direct result of estrogen exposure, and could constitute a useful marker of gonad feminisation and intersex condition.鱼类卵子发生（Fish oogenesis）的典型特征为每个生殖周期内卵母细胞发生大规模生长。该生长过程需要储备特定分子，例如核糖体RNA（ribosomal RNA, rRNA），以协助快速完成核糖体组装与蛋白质合成，保障新生胚胎发育进程的物质需求。卵母细胞中由转录因子3A（transcription factor 3A, Gtf3a）介导的大量5S rRNA表达，可作为鱼类暴露于外源性雌激素（xenoestrogens）后出现间性状态的标志物。本研究针对Gtf3a基因的演化开展了相关工作：在硬骨鱼（teleost）基因组中进行了计算机模拟分析，并在斑马鱼（Danio rerio）中开展了功能验证。对鱼类基因组的共线性分析（synteny analysis）显示，存在两个gtf3a旁系同源基因，二者大概率起源于硬骨鱼特异性基因组复制事件。功能分析结果表明，gtf3ab已演化成为一种特异性在卵母细胞中转录的基因，该现象在斑马鱼与尼罗罗非鱼（Oreochromis niloticus）中均有观测到。与之相反，gtf3aa则呈现广谱表达特征。此外，在斑马鱼胚胎中，gtf3aa的转录始于合子基因组激活阶段（约受精后8小时，8 hpf），而gtf3ab的转录仅在卵子发生起始阶段才会启动。当暴露于100 ng/L的17β-雌二醇（17β-estradiol）时，61日龄（受精后61天，61 dpf）完全雌性化的斑马鱼可检测到卵巢gtf3ab的转录；而经100 ng/L 17α-甲基睾酮（17α-methyltestosterone）处理实现雄性化的斑马鱼，则仅能检测到gtf3aa的转录。gtf3ab的性别相关转录模式与cyp19a1a一致，而与抗缪勒管激素（anti-Müllerian hormone, amh）及dmrt1（doublesex和mab-3相关转录因子1）的转录模式相反。在暴露于雌激素26天后尚未出现性腺发育迹象的幼鱼中，并未观测到gtf3ab的这种性别二态性转录模式。综上，gtf3ab的转录是卵母细胞分化的结果，而非雌激素暴露的直接效应，其可作为性腺雌性化与间性状态的有效标志物。