School of Marine Sciences, Ningbo University, Ningbo, Zhejiang 315211, China.
Aquat Toxicol. 2013 Sep 15;140-141:1-10. doi: 10.1016/j.aquatox.2013.05.004. Epub 2013 May 17.
Metallothioneins (MTs) possess a unique molecular structure that provides metal-binding and redox capabilities. These capabilities include the maintenance of metal equilibria that protect against heavy metals (especially cadmium) and oxidative damage. Past studies have focused on the function of MTs in vertebrates. However, the functions of MTs during spermiogenesis in invertebrates remain unclear. In order to investigate the function of MTs during spermiogenesis in Portunus trituberculatus, we used RT-PCR and RACE to identify two MT complete cDNA sequences in the total RNA from the P. trituberculatus testis. The 450 bp MT-1 cDNA consists of a 77 bp 5' untranslated region, a 196 bp 3' untranslated region, and a 177 bp open reading frame that encodes 58 amino acids including 19 cysteines. The 581 bp MT-2 cDNA consists of 73 bp 5' untranslated region, a 328 bp 3' untranslated region, and a 180 bp open reading frame that encodes 59 amino acids including 18 cysteines. MT-1 and MT-2 of P. trituberculatus more closely resemble invertebrate (especially crab) MT homologues than vertebrate MT homologues as indicated by protein alignment comparisons and phylogenetic tree analysis. MT-1 and MT-2 were detected in the heart, testis, muscle, hepatopancreas, and gill of P. trituberculatus by tissue expression analysis. In addition, MT-1 and MT-2 are present during the entire process of spermiogenesis in P. trituberculatus as indicated by H&E staining and in situ hybridization. MT-1 and MT-2 expression levels significantly increase after cadmium (Cd) exposure as measured by real-time quantitative PCR analysis. Therefore, we suggest that MT-1 and MT-2 perform important functions in spermiogenesis and testis detoxification in P. trituberculatus.
金属硫蛋白（MTs）具有独特的分子结构，提供金属结合和氧化还原能力。这些能力包括维持金属平衡，防止重金属（尤其是镉）和氧化损伤。过去的研究集中在 MTs 在脊椎动物中的功能。然而，在无脊椎动物的精子发生过程中 MTs 的功能仍不清楚。为了研究 MTs 在三疣梭子蟹精子发生过程中的功能，我们使用 RT-PCR 和 RACE 从三疣梭子蟹睾丸的总 RNA 中鉴定出两个 MT 完整 cDNA 序列。450 bp 的 MT-1 cDNA 由 77 bp 的 5'非翻译区、196 bp 的 3'非翻译区和 177 bp 的开放阅读框组成，该开放阅读框编码 58 个氨基酸，包括 19 个半胱氨酸。581 bp 的 MT-2 cDNA 由 73 bp 的 5'非翻译区、328 bp 的 3'非翻译区和 180 bp 的开放阅读框组成，该开放阅读框编码 59 个氨基酸，包括 18 个半胱氨酸。MT-1 和 MT-2 的三疣梭子蟹更类似于无脊椎动物（尤其是蟹）MT 同源物，而不是脊椎动物 MT 同源物，如蛋白质比对比较和系统发育树分析所示。通过组织表达分析，在三疣梭子蟹的心脏、睾丸、肌肉、肝胰腺和鳃中检测到 MT-1 和 MT-2。此外，通过 H&E 染色和原位杂交，MT-1 和 MT-2 存在于三疣梭子蟹精子发生的整个过程中。MT-1 和 MT-2 的表达水平在镉（Cd）暴露后显著增加，如实时定量 PCR 分析所示。因此，我们认为 MT-1 和 MT-2 在三疣梭子蟹的精子发生和睾丸解毒中发挥重要作用。
