Li Chuang-Ju, Zhou Li, Wang Yang, Hong Yun-Han, Gui Jian-Fang
State Key Laboratory of Freshwater Ecology and Biotechnology, Wuhan Center for Developmental Biology, Institute of Hydrobiology, Chinese Academy of Sciences, Graduate School of the Chinese Academy of Sciences, Wuhan 430072, China.
Mol Cell Endocrinol. 2005 Apr 15;233(1-2):33-46. doi: 10.1016/j.mce.2005.01.010.
A SMART cDNA plasmid library was constructed from protogyous greasy grouper (Epinephelus coioides) pituitary, and the full-length cDNAs of three gonadotropin (GTH) subunits common alpha, FSHbeta and LHbeta were cloned and sequenced from the library. The nucleotide sequences of common alpha, FSHbeta and LHbeta subunit cDNAs are 647, 594 and 574 bp in length, and encode for mature peptides of 94, 99 and 115 aa, respectively. High homology was observed by amino acid sequence alignment and identity comparison of the grouper mature peptides of common alpha, FSHbeta and LHbeta with that of other fishes. Phylogenetic tree analyses of the three GTH mature subunits revealed similar phylogeny relationships among the studied fish species. Three polyclonal antibodies were prepared from the in vitro expressed common alpha, FSHbeta and LHbeta mature proteins, respectively. Western blot analysis and immunofluoresence localization were performed on two typical stages of ovarian development stages in red-spotted grouper. Significant differences in protein expression levels of three gonadotropin subunits were revealed between the two ovarian development stages. In the individuals with resting ovary, common alpha was almost not detected in pituitaries, and FSHbeta and LHbeta expression levels were very low. While in the individuals with developing ovary, the expression of all three gonadotropin subunits reached to a high level. Immunofluoresence localization indicated that the grouper FSHbeta cells mainly distributed in the middle area of PPD, while the LHbeta cells distributed more widely, including in the area similar to the FSHbeta cells and at the external periphery of pituitary near to the PI side. The common alpha might be expressed in both FSHbeta and LHbeta cells. Double immunofluoresence localization further demonstrated FSHbeta and LHbeta expression in distinct cells in the PPD area, although the FSHbeta and LHbeta cells were detected in the identical area of PPD.
从早生的斜带石斑鱼（Epinephelus coioides）垂体构建了一个SMART cDNA质粒文库，并从该文库中克隆并测序了三种促性腺激素（GTH）亚基共同α、FSHβ和LHβ的全长cDNA。共同α、FSHβ和LHβ亚基cDNA的核苷酸序列长度分别为647、594和574 bp，分别编码94、99和115个氨基酸的成熟肽。通过氨基酸序列比对以及斜带石斑鱼共同α、FSHβ和LHβ成熟肽与其他鱼类成熟肽的同一性比较，发现具有高度同源性。对三种GTH成熟亚基的系统发育树分析揭示了所研究鱼类物种之间相似的系统发育关系。分别从体外表达的共同α、FSHβ和LHβ成熟蛋白制备了三种多克隆抗体。对红斑石斑鱼卵巢发育的两个典型阶段进行了蛋白质免疫印迹分析和免疫荧光定位。揭示了两个卵巢发育阶段三种促性腺激素亚基的蛋白质表达水平存在显著差异。在卵巢静止的个体中，垂体中几乎检测不到共同α，FSHβ和LHβ的表达水平非常低。而在卵巢发育的个体中，所有三种促性腺激素亚基的表达都达到高水平。免疫荧光定位表明，石斑鱼FSHβ细胞主要分布在PPD的中间区域，而LHβ细胞分布更广泛，包括在与FSHβ细胞相似的区域以及靠近PI侧的垂体外周。共同α可能在FSHβ和LHβ细胞中均有表达。双重免疫荧光定位进一步证明了PPD区域中不同细胞中FSHβ和LHβ的表达，尽管在PPD的相同区域检测到了FSHβ和LHβ细胞。
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