Japanese Eel Follicle-Stimulating Hormone (Fsh) and Luteinizing Hormone (Lh): Production of Biologically Active Recombinant Fsh and Lh by Drosophila S2 Cells and Their Differential Actions on the Reproductive Biology
Japanese Eel Follicle-Stimulating Hormone (Fsh) and Luteinizing Hormone (Lh): Production of Biologically Active Recombinant Fsh and Lh by Drosophila S2 Cells and Their Differential Actions on the Reproductive Biology
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DOI：
10.1095/biolreprod.108.070052
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发表时间：
2008-11-01
影响因子：
3.6
通讯作者：
Yamauchi, Kohei
中科院分区：
文献类型：
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作者：
Kazeto, Yukinori;Kohara, Mayuko;Yamauchi, Kohei
两种促性腺激素（Gths），促卵泡激素（FSH）和促黄体激素（Lh），控制脊椎动物（包括硬骨鱼）的性腺类固醇和配子发生。在这里，我们报告的生产生物活性的重组FSH（Rec-FSH）和Lh（Rec-Lh）在日本鳗鱼使用果蝇S2细胞。组成Gth的三个子单元，即，糖蛋白激素α多肽（Cga）、促卵泡激素β多肽（Fshb）和促黄体生成激素β多肽（Lhb）最初是独立产生的，并被证明是糖基化的并作为成熟肽分泌。每个β亚基，沿着其Cga，同时共表达以产生异二聚体rec-FSH和rec-Lh，其随后被高度纯化。在各种体外测定中证明了rec-Gth的生物活性。rec-Gth差异激活其受体，导致11-酮睾酮（11-ketosterone，11 KT）分泌增加，未成熟睾丸中类固醇生成酶基因表达的差异改变，以及体外精子发生的完整过程的诱导。这些数据强烈表明，FSH和LH差异发挥重要作用的生殖生理的日本鳗鱼。相比之下，这些rec-Gth在体内给药时在性腺中表现出很小的活性。体外和体内生物活性之间的这种差异可能是由于S2细胞中糖基化的定性性质，这导致重组蛋白在体内降解。碳水化合物部分的这些差异需要得到阐明和改善。
Two gonadotropins (Gths), follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh), control gonadal steroidogenesis and gametogenesis in vertebrates, including teleost fish. Here, we report on the production of biologically active recombinant Fsh (rec-Fsh) and Lh (rec-Lh) in Japanese eel using Drosophila S2 cells. The three subunits composing Gths, i.e., glycoprotein hormone, alpha polypeptide (Cga), follicle-stimulating hormone, beta polypeptide (Fshb), and luteinizing hormone, beta polypeptide (Lhb), were at first independently produced and were proven to be glycosylated and secreted as the mature peptides. Each beta subunit, along with its Cga, was simultaneously coexpressed to produce heterodimeric rec-Fsh and rec-Lh that were subsequently highly purified. The biological activity of rec-Gths was demonstrated in various in vitro assays. The rec-Gths differentially activated their receptors, which resulted in an increase in 11-ketotestosterone (11KT) secretion, a differential alteration of gene expression of steroidogenic enzymes in immature testis, and the induction of the complete process of spermatogenesis in vitro. The data strongly suggest that Fsh and Lh differentially play important roles in the reproductive physiology of the Japanese eel. By contrast, these rec-Gths exhibited little activity in the gonad when administered in vivo. This difference between in vitro and in vivo bioactivity is probably due to the qualitative nature of glycosylation in S2 cells, which resulted in degradation of the recombinant protein in vivo. These differences in the carbohydrate moieties need to be elucidated and ameliorated.