Vafopoulou Xanthe, Steel Colin G H, Terry Katherine L
Biology Department, York University, 4700 Keele St., Toronto, Ontario, Canada, M3J 1P3.
J Insect Physiol. 2005 Jan;51(1):27-38. doi: 10.1016/j.jinsphys.2004.11.001.
The presence of ecdysteroid receptor (EcR) in various tissues was studied throughout larval-adult development of the blood-sucking bug, Rhodnius prolixus, using an antibody to EcR that recognizes all isoforms. On Western blots, the antibody recognizes three peptides of approximate molecular masses of 70, 68 and 64 kDa, from epidermis and fat body of developing larvae, which contain high levels of haemolymph ecdysteroids. These peptides are absent from both unfed larvae and adults, which are devoid of ecdysteroids. In vitro treatment of epidermis and fat body from unfed larvae with 20E induces the appearance of all three EcR immunoreactive peptides. The stage-specific appearance and 20E inducibility of the peptides implies that they represent the native EcR(s) of Rhodnius. Confocal fluorescence analysis using this antibody revealed a great diversity of temporal profiles of EcR in various tissues during development. Developmental profiles of EcR were examined in abdominal epidermis, fat body, spermatocytes, brain (including the medial neurosecretory cells), prothoracic glands (PGs), rectal epithelium and Malpighian tubules. EcR fluorescence was confined to the nuclei in close association with chromatin. EcR was absent from tissues of unfed larvae or adults, supporting the results from Western blots. Different tissues develop EcR at different developmental times and in the presence of radically different concentrations of haemolymph ecdysteroids, retain EcR for different lengths of time and lose EcR at different concentrations of ecdysteroids. These results suggest that each tissue possesses a distinctive response mechanism to ecdysteroids. An exception to this, are the PGs, which exhibited no EcR fluorescence at any time during development.
利用一种能识别所有异构体的蜕皮激素受体(EcR)抗体,研究了吸血蝽象(Rhodnius prolixus)幼虫到成虫发育全过程中不同组织内EcR的存在情况。在蛋白质免疫印迹分析中,该抗体识别出分子量约为70、68和64 kDa的三种肽段,它们来自发育中幼虫的表皮和脂肪体,这些组织中含有高水平的血淋巴蜕皮激素。未取食的幼虫和成虫体内均不存在这些肽段,因为它们体内没有蜕皮激素。用20E对未取食幼虫的表皮和脂肪体进行体外处理,可诱导出所有三种EcR免疫反应性肽段的出现。这些肽段的阶段特异性出现以及20E诱导性表明它们代表了猎蝽的天然EcR。使用该抗体进行的共聚焦荧光分析揭示了发育过程中不同组织内EcR在时间分布上的巨大差异。对腹部表皮、脂肪体、精母细胞、脑(包括内侧神经分泌细胞)、前胸腺(PGs)、直肠上皮和马氏管中的EcR发育分布情况进行了研究。EcR荧光局限于与染色质紧密相关的细胞核中。未取食幼虫或成虫的组织中不存在EcR,这与蛋白质免疫印迹分析的结果一致。不同组织在不同发育时间、在血淋巴蜕皮激素浓度差异极大的情况下发育出EcR,EcR保留的时间不同,且在不同蜕皮激素浓度下失去EcR。这些结果表明,每个组织对蜕皮激素都具有独特的反应机制。例外的是前胸腺,其在发育过程中的任何时候都未表现出EcR荧光。