Sunderman F W, Grbac-Ivankovic S, Plowman M R, Davis M
Department of Laboratory Medicine, University of Connecticut School of Medicine, Farmington, USA.
Mol Reprod Dev. 1996 Apr;43(4):444-51. doi: 10.1002/(SICI)1098-2795(199604)43:4<444::AID-MRD6>3.0.CO;2-P.
The localization of metallothionein in control and Zn-exposed embryos of Xenopus laevis was studied by whole-mount immunohistochemical staining. The embryos were grown according to the FETAX (Frog Embryo Teratogenesis Assay: Xenopus) protocol from N/F stage 8 to stage 47, with or without addition of ZnCl2 (300 microM) to the medium. At stages 27, 38, 42, 45 and 47, control and Zn-exposed embryos were fixed in buffered formalin, and whole mounts were stained by an immunoperoxidase technique, using monoclonal murine antibody to equine metallothionein. Staining of metallothionein was evident in myotomal cell nuclei of developing somites by stage 27, stomatodeum, oropharynx, and gills by stage 38, developing kidneys (mesonephros) by stage 45, and liver by stage 47. The staining of metallothionein at these sites was more intense in Zn-exposed embryos than controls. The central nervous system (especially the spinal cord) and the yolk mass were faintly stained for metallothionein in controls and Zn-exposed embryos. Staining of metallothionein in myotomal cell nuclei was most prominent at stage 38, diminished at stages 42 and 45, and practically disappeared by stage 47. This is the first report that metallothionein is expressed in myotomal cell nuclei of Xenopus embryos during normal somitogenesis and becomes increased when the embryos are exposed to teratogenic levels of Zn2+.
通过整装免疫组织化学染色研究了非洲爪蟾对照胚胎和锌暴露胚胎中金属硫蛋白的定位。胚胎按照FETAX（青蛙胚胎致畸试验：非洲爪蟾）方案从N/F阶段8生长到47阶段，培养基中添加或不添加ZnCl2（300 microM）。在第27、38、42、45和47阶段，将对照胚胎和锌暴露胚胎固定在缓冲福尔马林中，并用免疫过氧化物酶技术对整装标本进行染色，使用抗马金属硫蛋白的单克隆鼠抗体。到第27阶段，金属硫蛋白在发育中的体节的肌节细胞核中明显染色，到第38阶段，在口凹、口咽和鳃中染色，到第45阶段，在发育中的肾脏（中肾）中染色，到第47阶段，在肝脏中染色。锌暴露胚胎中这些部位的金属硫蛋白染色比对照胚胎更强烈。在对照胚胎和锌暴露胚胎中，中枢神经系统（尤其是脊髓）和卵黄团对金属硫蛋白的染色较淡。金属硫蛋白在肌节细胞核中的染色在第38阶段最为突出，在第42和45阶段减弱，到第47阶段几乎消失。这是关于金属硫蛋白在非洲爪蟾胚胎正常体节发生过程中在肌节细胞核中表达，并且当胚胎暴露于致畸水平的Zn2+时表达增加的首次报道。
