乙炔基雌二醇（EE2）对雄性黄颡鱼GtH 3个亚基基因表达的影响

摘要

In this paper, we investigated how 17α-Ethynylestradiol (EE2) regulated the expression of three gonadotropin subunits in malePelteobagrus fulvidraco. We cloned the full-length cDNA sequences of two subunits of gonadotropin hormone (FSHβ andLHβ) from the pituitary ofPelteobagrus fulvidracoby using RACE. We explored the tissue-specific and the seasonal pattern of expression of the genes. MalePelteobagrus fulvidraco (2 years old) were exposed to EE2 (100 ng/L) for 28 days. We found that the length ofFSHβ cDNA sequence was 528 bp and it contained a 399-bp open reading frame (ORF) which encoded a precursor protein of 132 amino acids (aa). The length ofLHβ cDNA sequence was 870 bp and it contained a 417 bp ORF that encoded a precursor protein of 138 aa. The sequence analysis showed that FSHβ had a predicted signal peptide of 17 aa, 2 N-glycosylation sites, and 13 cysteine residues. LHβ had a pre-dicted signal peptide of 18 aa, 1 N-glycosylation site, and 12 cysteine residues. The phylogenetic analysis showed that FSHβ and LHβ were closely related to other Siluriformes species. The tissue expression analysis suggested that all the gonadotropin subunits were expressed specifically in pituitary. The seasonal pattern of expression in both genders was thatGtHαandLHβmRNA peaked in May and then decreased gradually, and the expression ofFSHβin female also peaked in May but the expression in male remained unchanged. We applied semi-quantitative RT-PCR and demonstrated that the expression of the three gonadotropin subunits was dramatically suppressed by EE2. We speculated that EE2 might reduce the generation of FSH and LH in the pituitary of malePelteobagrus fulvidraco, which would probably inhibit spermatogenesis and the maturation of sperm and spermiation, and consequently impair the testicular develop-ment and reproduction.%为研究乙炔基雌二醇(EE2)是否能影响雄性黄颡鱼(Pelteobagrus fulvidraco)垂体中促性腺激素3个亚基基因的表达,从而干扰 FSH 和 LH 的分泌,研究采用末端快速扩增(RACE)的方法在黄颡鱼垂体中克隆了促性腺激素的2个β亚基(FSHβ和LHβ)的全长cDNA,对其组织表达模式和雌雄性垂体中的季节表达模式进行了研究；另外,研究还用100 ng/L的EE2对雄性黄颡鱼(2龄)进行了28d的暴露处理。结果发现,黄颡鱼FSHβ cDNA全长528 bp, ORF为399 bp,编码132个氨基酸；LHβ全长为870 bp, ORF为417 bp,编码138个氨基酸。序列分析结果表明,黄颡鱼FSHβ含有一个17氨基酸的信号肽,2个保守的N-糖基化位点和13个半胱氨酸残基,而LHβ含有一个18个氨基酸的信号肽,1个N-糖基化位点和12个半胱氨酸残基,与其他鲶形目鱼类极其相似。进化分析显示,黄颡鱼FSHβ和LHβ与鲶形目的鱼类进化关系较近。组织分布结果发现,黄颡鱼3亚基均仅在垂体中表达。季节表达模式结果表明,雌雄黄颡鱼GtHα和LHβ表达水平在5月份左右达到最高,随后降低；FSHβ在雌性的表达模式与GtHα和LHβ相同,而在雄性, FSHβ的表达没有明显变化。半定量RT-PCR结果显示,100 ng/L的EE2能显著抑制黄颡鱼促性腺激素3个亚基基因的表达。研究认为, EE2抑制黄颡鱼雄性垂体FSH和LH的分泌,可能阻碍其正常的精子发生、精子成熟和排精过程,从而影响其正常的繁殖和发育。