Hellqvist Anna, Bornestaf Cecilia, Borg Bertil, Schmitz Monika
Department of Zoology, Stockholm University, S-106 91 Stockholm, Sweden.
Gen Comp Endocrinol. 2004 Jan 15;135(2):167-74. doi: 10.1016/j.ygcen.2003.09.003.
The aim of this study was to characterize the FSH-beta and LH-beta subunit in the three-spined stickleback, a fish used extensively in experimental studies, and to use the cloned cDNAs as probes for measuring FSH-beta and LH-beta mRNA expression in sticklebacks treated with different photoperiods and temperatures. A first strand cDNA was prepared from 10 pituitaries from male sticklebacks, and cDNA fragments were amplified by PCR using degenerated primers based on highly conserved regions of known teleost FSH-beta and LH-beta cDNA sequences. To obtain full-length cDNAs of FSH-beta and LH-beta, RACE amplifications were performed. The cDNA of FSH-beta was 540 bp long, encoding a protein of 122 amino acids and LH-beta cDNA was 568 bp long, encoding a protein of 148 amino acids. Of gonadotropin (GTH) beta-subunits published so far, those most similar to stickleback GTHs are found among other percomorph fishes, with amino acid similarities of 46-55% for FSH-beta and 75-77% for LH-beta subunits.The cloned cDNAs were used as probes to analyze LH and FSH mRNA expression in pituitaries from sticklebacks exposed to different photoperiods and temperatures. Two hundred males were divided into four groups and kept under different photoperiods and temperatures; light:dark (LD) 16:8, 20 degrees C; LD 16:8, 7 degrees C; LD 8:16, 20 degrees C, and LD 8:16, 7 degrees C. Red breeding colors and a marked kidney hypertrophy, androgen-dependent male secondary sexual characters in the stickleback, appeared in the group kept under LD 16:8 at 20 degrees C, but not in the other groups. The kidney epithelium height (KEH) was significantly lower in the LD 8:16, 20 degrees C group than in all other groups, and this was also the only group with active spermatogenesis. The LD 8:16 20 degrees C had significantly lower expression of both FSH-beta and LH-beta than all other groups. Thus, both GTHs displayed their lowest expression when spermatogenesis was active and when low KEH indicated that androgens levels were at their lowest.
本研究的目的是对三刺鱼（一种在实验研究中广泛使用的鱼类）的促卵泡激素β亚基（FSH-β）和促黄体生成素β亚基（LH-β）进行特征分析，并使用克隆的cDNA作为探针，来测定在不同光周期和温度处理下的三刺鱼中FSH-β和LH-β mRNA的表达。从10条雄性三刺鱼的垂体中制备第一链cDNA，并使用基于已知硬骨鱼FSH-β和LH-β cDNA序列高度保守区域的简并引物，通过PCR扩增cDNA片段。为了获得FSH-β和LH-β的全长cDNA，进行了RACE扩增。FSH-β的cDNA长540 bp，编码一个122个氨基酸的蛋白质，LH-β的cDNA长568 bp，编码一个148个氨基酸的蛋白质。在迄今已发表的促性腺激素（GTH）β亚基中，与三刺鱼GTH最相似的是在其他鲈形目鱼类中发现的，FSH-β亚基的氨基酸相似性为46-55%，LH-β亚基的氨基酸相似性为75-77%。克隆的cDNA被用作探针，以分析暴露于不同光周期和温度下的三刺鱼垂体中LH和FSH mRNA的表达。200条雄性三刺鱼被分成四组，并饲养在不同的光周期和温度条件下：光照：黑暗（LD）16:8，20℃；LD 16:8，7℃；LD 8:16，20℃，以及LD 8:16，7℃。红色繁殖体色和明显的肾脏肥大（三刺鱼中雄激素依赖性的雄性第二性征）出现在20℃下饲养在LD 16:8条件下的组中，但在其他组中未出现。在LD 8:16，20℃组中，肾上皮高度（KEH）显著低于所有其他组，并且这也是唯一具有活跃精子发生的组。在LD 8:16，20℃组中，FSH-β和LH-β的表达均显著低于所有其他组。因此，当精子发生活跃且低KEH表明雄激素水平处于最低时，两种GTH均表现出最低表达。
