甲状腺激素受体在扬子鳄卵巢中的免疫组织化学定位和不同繁殖时期的mRNA表达水平变化检测

Immunohistochemical localization of thyroid hormone receptor and detection for variation of m RNA level in different reproductive periods in the ovary of the Chinese alligator

目的研究扬子鳄甲状腺激素受体(thyroid hormone receptor,TR)基因的系统进化关系和在卵巢组织中的定位,分析成年扬子鳄在不同繁殖活动期卵巢组织中甲状腺激素受体的动态变化规律。方法通过Clustal W软件对扬子鳄和其它脊椎动物TRs氨基酸序列进行序列比对,使用MEGA 6.0软件构建NJ系统发育树;应用q RT-PCR方法检测TRα和TRβm RNA在不同繁殖时期卵巢组织中的表达水平差异;应用免疫组织化学法对扬子鳄卵巢卵泡中TR进行组织定位。结果系统发育分析显示扬子鳄与鸟类亲缘关系较近;扬子鳄卵巢中TR的m RNA表达水平在繁殖期最高,在繁殖前期和繁殖后期相对较低;免疫组织化学染色显示,TR阳性反应主要位于卵泡的颗粒层细胞、卵泡膜、卵母细胞质和卵母细胞核中,其中颗粒层细胞阳性反应最明显。结论 TR基因的进化时间较早,因此序列保守性较高;TRα和TRβ对扬子鳄卵泡发育成熟具有重要作用;颗粒层细胞是TRs的功能作用主要位点。

Objective To investigate thyroid hormone receptor(TR)localization in the ovarian tissue,and the phylogenetic relationships of TR genes in Chinese alligators;also analyze the variation of their expression in the ovary at different reproductive stages in adult Chinese alligators.Methods The TR amino acid sequences of the Chinese alligator and other vertebrates were compared by Clustal W software.The NJ phylogenetic tree was built by MEGA6.0software.The variation of the mRNA levels of TRαand TRβin the ovary at different reproductive stages was detected by qRT-PCR.Immunohistochemistry was used to locate TR in the ovarian follicles of Chinese alligators.Results The phylogenetic analysis showed that the Chinese alligator had a close relationship with birds in the phylogeny tree.The TR mRNA level in the ovary of the Chinese alligator was the highest in the breeding season and relatively lower in the pre-reproductive and post-reproductive periods.Immunohistochemical staining results showed that TR was mainly located in the granular layer,theca follicle,oocyte cytoplasm and nuclei of the follicle,and the reactivity was the most remarkable in the granular layer cells.Conclusion TR genes evolved early in the phylogeny tree,and their sequences are highly conserved.TRαand TRβplay an important role in the development and maturation of the follicle of Chinese alligators,and function mainly in the granular layer cells.