Yamamoto K, Toyoda F, Tanaka S, Hayashi H, Kikuyama S
Department of Biology, School of Education, Waseda University, Tokyo, Japan.
Gen Comp Endocrinol. 1996 Dec;104(3):356-63. doi: 10.1006/gcen.1996.0181.
A specific and sensitive radioimmunoassay (RIA) was developed for the measurement of sodefrin, a female-attracting decapeptide pheromone of the red-bellied newt, Cynops pyrrhogaster. Sodefrin synthesized according to the amino acid sequence of native sodefrin, isolated from the abdominal glands of the cloaca of the male newt, was used as a reference standard. An antiserum to sodefrin was produced by immunizing a rabbit with synthetic sodefrin that was extended on its C-terminus with Cys coupled to hemocyanin. For the radioligand, sodefrin N-terminally extended with Tyr was used. An aqueous extract of the abdominal glands of C. pyrrhogaster produced a displacement curve parallel to the sodefrin standard, whereas that from the sword-tailed newt (Cynops ensicauda) showed no inhibition of binding in this RIA. The sensitivity of the RIA was 30.5 +/- 3.4 pg/100 microliters assay buffer. Intraassay and interassay coefficients of variation were 1.6 and 4.5%, respectively. The RIA was used to determine sodefrin levels in the abdominal gland of the male newt. Hypophysectomy and castration greatly reduced the sodefrin content. Administration of testosterone propionate (TP) to the hypophysectomized and castrated newt increased the pheromone content in the abdominal gland. A combination of prolactin (PRL) and TP elevated the sodefrin content markedly, while PRL alone scarcely affected it. Immunoreactive sodefrin was observed in the epithelial cells of the abdominal gland of both hormone-treated and saline-injected newts. Among them, groups treated with PRL plus TP and TP alone exhibited strong immunoreactivity in their abdominal gland compared with PRL-treated and saline-injected groups.
开发了一种特异性和敏感性均高的放射免疫分析法（RIA），用于测量红腹蝾螈（Cynops pyrrhogaster）的雌性吸引十肽信息素——精素。根据从雄性蝾螈泄殖腔腹腺分离出的天然精素的氨基酸序列合成的精素，用作参考标准品。通过用在其C末端与偶联血蓝蛋白的半胱氨酸延伸的合成精素免疫兔子，制备了抗精素血清。对于放射性配体，使用在N末端用酪氨酸延伸的精素。红腹蝾螈腹腺的水提取物产生了与精素标准品平行的置换曲线，而剑尾蝾螈（Cynops ensicauda）的水提取物在该RIA中未显示结合抑制。该RIA的灵敏度为30.5±3.4 pg/100微升测定缓冲液。批内和批间变异系数分别为1.6%和4.5%。该RIA用于测定雄性蝾螈腹腺中的精素水平。垂体切除和去势大大降低了精素含量。给垂体切除和去势的蝾螈注射丙酸睾酮（TP）可增加腹腺中的信息素含量。催乳素（PRL）和TP的组合显著提高了精素含量，而单独使用PRL几乎没有影响。在激素处理和注射生理盐水的蝾螈的腹腺上皮细胞中均观察到免疫反应性精素。其中，与PRL处理组和注射生理盐水组相比，PRL加TP处理组和单独TP处理组在其腹腺中表现出强烈的免疫反应性。
