State Key Laboratory of Biocontrol, Institute of Aquatic Economic Animals and Guangdong Provincial Key Laboratory for Aquatic Economic Animals, Sun Yat-Sen University, Guangzhou 510275, China.
Gen Comp Endocrinol. 2012 Sep 1;178(2):237-49. doi: 10.1016/j.ygcen.2012.05.009. Epub 2012 Jun 7.
FSH is a key regulator of steroidogenesis and gonadal growth in teleosts. However, function of FSH is elusive in grouper due to the lack of purified and native FSH. In the present study, we reported production of bioactive orange-spotted grouper (Epinephelus coioides) FSH in dimer form and single-chain form by Pichia pastoris. Dimer form of recombinant grouper FSH (rgFSHba) was accomplished by co-expressing mature FSHb-subunit and a-subunit genes. Fusion of mature FSHb-subunit and a-subunit genes together linking with a polypeptide (4×(Gly-Ser)-Gly-Thr) gene generated single-chain form of recombinant grouper FSH (rgFSHb-a). Recombinant grouper common α-subunit (rgCga) and FSHb-subunit (rgFSHb) were also separately produced. Recombinant proteins were verified by Western blot and mass spectrometry assays, and characterized by deglycosylation analysis. Deglycosylation assay suggested that glycosylation of recombinant FSH mainly occurred on common a-subunit. Bioactivities of recombinant proteins were initially evaluated by activating grouper FSH receptor, and further demonstrated by incubating ovarian fragments of adult grouper and intraperitoneal injection in juvenile female grouper. Two forms of recombinant FSH presented similar biological activities of activating FSH receptor and stimulating in vitro testosterone (T) and estradiol-17β (E2) secretion, though the dimer form functioned slightly weaker than the single-chain form. However, injections of rgFSHb-a or rgFSHba could significantly increase serum T and E2 levels, induce early ovarian development, reduce hypothalamic gnrh1 mRNA level, and increase hypothalamic cyp19a1b mRNA level. Data in this study suggested that recombinant gonadotropin could be produced in dimer form or single-chain form by P. pastoris, and FSH could regulate steroidogenesis and early ovarian development in juvenile grouper.
FSH 是硬骨鱼类类固醇生成和性腺生长的关键调节剂。然而，由于缺乏纯化和天然的 FSH，在石斑鱼中 FSH 的功能仍不清楚。在本研究中，我们通过毕赤酵母报告了二聚体形式和单链形式的生物活性橙色斑点石斑鱼（Epinephelus coioides）FSH 的产生。重组石斑鱼 FSH（rgFSHba）的二聚体形式通过共表达成熟 FSHb-亚基和 a-亚基基因来完成。成熟 FSHb-亚基和 a-亚基基因融合在一起，连接一个多肽（4×（甘氨酸-丝氨酸）-甘氨酸-苏氨酸）基因，产生重组石斑鱼 FSH 的单链形式（rgFSHb-a）。重组石斑鱼 common α-亚基（rgCga）和 FSHb-亚基（rgFSHb）也分别进行了表达。通过 Western blot 和质谱分析验证重组蛋白，并通过糖基化分析进行表征。糖基化分析表明，重组 FSH 的糖基化主要发生在 common a-亚基上。通过激活石斑鱼 FSH 受体初步评估了重组蛋白的生物活性，并通过在成年石斑鱼卵巢片段孵育和幼年雌性石斑鱼腹腔内注射进一步证实。两种形式的重组 FSH 表现出相似的激活 FSH 受体和刺激体外睾酮（T）和雌二醇-17β（E2）分泌的生物学活性，尽管二聚体形式的功能略弱于单链形式。然而，rgFSHb-a 或 rgFSHba 的注射可显著增加血清 T 和 E2 水平，诱导早期卵巢发育，降低下丘脑 gnrh1 mRNA 水平，并增加下丘脑 cyp19a1b mRNA 水平。本研究的数据表明，重组促性腺激素可以通过毕赤酵母以二聚体形式或单链形式产生，FSH 可以调节幼鱼石斑鱼的类固醇生成和早期卵巢发育。
