Aizen Joseph, Kasuto Harel, Levavi-Sivan Berta
Department of Animal Sciences, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University, P.O. Box 12, Rehovot 76100, Israel.
Gen Comp Endocrinol. 2007 Aug-Sep;153(1-3):323-32. doi: 10.1016/j.ygcen.2007.04.004. Epub 2007 Apr 11.
We recently produced Oreochromis niloticus recombinant LH and FSH as single-chain polypeptides in the methylotrophic yeast Pichia pastoris. Glycoprotein subunit alpha was joined with tilapia (t) LHbeta or tFSHbeta mature protein-coding sequences to form a fusion gene that encodes a ;;tethered" polypeptide, in which the gonadotropin beta-subunit forms the N-terminal part and the alpha-subunit forms the C-terminal part. Recombinant (r) gonadotropins were used to develop specific and homologous competitive ELISAs for measurements of FSH and LH in the plasma and pituitary of tilapia, using primary antibodies against rtLHbeta or rtFSHbeta, respectively, and rtLHbetaalpha or rtFSHbetaalpha for the standard curves. The wells were coated with either rtLHbeta (2ng/ml) or rtFSHbeta (0.5ng/well), and the final concentrations of the antisera were 1:5000 (for tLH) or 1:50,000 (for tFSH). The sensitivity of the assay was 15.84pg/ml for tLH and 0.24pg/ml for tFSH measurements in the plasma, whereas for the measurements in the pituitary, the sensitivity was 2.43ng/ml and 1.52ng/ml for tLH and tFSH, respectively. The standard curves for tFSH and tLH paralleled those of serially diluted pituitary extracts of other cichlids, as well as of serially diluted pituitary extract of seabream, European seabass and hybrid bass. We examined plasma tFSH and tLH levels in the course of one reproductive cycle, between two successive spawnings, in three individual tilapia females. Plasma levels of both FSH and LH increased during the second day after the eggs had been removed, probably related to the vitellogenic phase. LH levels increased toward spawning, which occurred on the 11th day. FSH levels also increased on day of cycle, probably due to recruitment of a new generation of follicles for the successive spawning. The development of specific ELISAs using recombinant gonadotropins is expected to advance the study of the distinct functions of each of these important hormones.
我们最近在甲基营养型酵母毕赤酵母中成功生产了尼罗罗非鱼重组促黄体生成素（LH）和促卵泡生成素（FSH），它们均为单链多肽。糖蛋白亚基α与罗非鱼（t）LHβ或tFSHβ成熟蛋白编码序列相连，形成一个融合基因，该基因编码一种“拴系”多肽，其中促性腺激素β亚基构成N端部分，α亚基构成C端部分。重组（r）促性腺激素被用于开发特异性和同源竞争性酶联免疫吸附测定法（ELISA），以测量罗非鱼血浆和垂体中的FSH和LH，分别使用针对rtLHβ或rtFSHβ的一抗，以及rtLHβα或rtFSHβα作为标准曲线。酶标板用rtLHβ（2ng/ml）或rtFSHβ（0.5ng/孔）包被，抗血清的终浓度为1:5000（用于tLH）或1:50,000（用于tFSH）。该测定法对血浆中tLH的检测灵敏度为15.84pg/ml，对tFSH的检测灵敏度为0.24pg/ml，而对于垂体中的检测，tLH和tFSH的灵敏度分别为2.43ng/ml和1.52ng/ml。tFSH和tLH的标准曲线与其他丽鱼科鱼类垂体提取物系列稀释液以及黑鲷、欧洲鲈鱼和杂交鲈鱼垂体提取物系列稀释液的标准曲线平行。我们检测了三条罗非鱼雌鱼在一个生殖周期内、两次连续产卵之间血浆中tFSH和tLH的水平。在卵子被取出后的第二天，血浆中FSH和LH的水平均升高，这可能与卵黄生成期有关。LH水平在第11天产卵时升高。在周期的某一天FSH水平也升高，这可能是由于为连续产卵募集了新一代卵泡。使用重组促性腺激素开发特异性ELISA有望推动对这些重要激素各自独特功能的研究。
