Fisher B R, Heredia D J, Brown K M
Health Sciences Branch, Food and Drug Administration, Rockville, Maryland 20857, USA.
Teratology. 1995 Aug;52(2):90-100. doi: 10.1002/tera.1420520205.
Previous studies have demonstrated that heat exposure on gestation day 10 (GD10) resulted in disrupted somite development in rat embryos 24 hr after exposure and in thoracic skeletal malformations in neonatal rats examined 3 days postpartum. The production of abnormal somites was correlated with the location of skeletal elements that developed from the affected somites. Heat has also been shown to induce changes in genetic expression whereby new proteins are synthesized and the expression of constituent proteins may be repressed. In the present study, heat-induced alterations in protein synthesis during rat organogenesis that may be associated with previously observed malformation was investigated. GD10 rat embryos were exposed in utero to a heat treatment previously demonstrated to produce skeletal malformations; maternal core temperature was raised and maintained at 42-42.4 degrees C for 5 min. In addition, explanted GD10 embryos were cultured in vitro and exposed to temperatures of 42-42.5 degrees C for 15 min. At various times postexposure, embryos were labeled with 35S-methionine and processed for SDS-PAGE. In both in vivo and in vitro heat-treated embryos, a transient enhanced de novo synthesis of 70- and 90-kD proteins was observed 1-8 hr after exposure. Actinomycin D studies were conducted to determine whether transcription of new mRNA was required for the enhanced synthesis of the 70- and 90-kD proteins in heat-treated embryos. Results from these studies demonstrated that the expression of these proteins was transcriptionally regulated. The 70-kD protein was identified, using Western blot analysis, as a eukaryotic inducible stress protein (hsp72), and the presence of this protein was detected between 2 and 27 hr post-treatment. Immunohistochemical results indicated that following heat shock, hsp72 accumulates in the neuroectodermal tissues of the embryos. The data demonstrate that although heat-induced expression and accumulation of the hsp72 precedes aberrant somite morphology, the lack of hsp72 accumulation in the somite mesoderm may explain the sensitivity of this tissue to heat.
先前的研究表明，在妊娠第10天（GD10）受热，会导致大鼠胚胎在受热后24小时体节发育紊乱，并在产后3天检查发现新生大鼠出现胸廓骨骼畸形。异常体节的产生与由受影响体节发育而来的骨骼元素的位置相关。研究还表明，热会诱导基因表达发生变化，从而合成新的蛋白质，同时组成蛋白质的表达可能受到抑制。在本研究中，对大鼠器官形成过程中热诱导的蛋白质合成变化进行了研究，这些变化可能与先前观察到的畸形有关。GD10大鼠胚胎在子宫内接受先前已证明会导致骨骼畸形的热处理；母体核心体温升高并维持在42 - 42.4摄氏度5分钟。此外，将分离出的GD10胚胎进行体外培养，并暴露于42 - 42.5摄氏度15分钟。在暴露后的不同时间，用35S - 甲硫氨酸标记胚胎，并进行SDS - PAGE处理。在体内和体外受热处理的胚胎中，暴露后1 - 8小时均观察到70kD和90kD蛋白质的瞬时从头合成增强。进行放线菌素D研究以确定热处理胚胎中70kD和90kD蛋白质合成增强是否需要新mRNA的转录。这些研究结果表明，这些蛋白质的表达受转录调控。通过蛋白质免疫印迹分析鉴定出70kD蛋白质为真核诱导应激蛋白（hsp72），并且在处理后2至27小时检测到该蛋白质的存在。免疫组织化学结果表明，热休克后，hsp72在胚胎的神经外胚层组织中积累。数据表明，尽管热诱导的hsp72表达和积累先于异常体节形态出现，但体节中胚层中缺乏hsp72积累可能解释了该组织对热的敏感性。
