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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., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Abstract: The Piwi genes have an important role in stem cell development, gametogenesis and RNA interference in diverse organisms. So far, most of the studies have focused on the function of Piwis in vertebrates, but their function during spermiogenesis in invertebrates still remains largely unclear. In order to investigate the function of Piwis during spermiogenesis in the crab Portunus trituberculatus, we use RT-PCR and RACE to identify three Piwi complete cDNA sequences from the total RNA of the testis in P. trituberculatus. The deduced amino acid sequences of P. trituberculatus Piwi-1, Piwi-2 and Piwi-3 showed that each contains a well-conserved PAZ domain and PIWI domain. RT-PCR analyzed the tissue expression pattern of P. trituberculatus Piwi-1, Piwi-2 and Piwi-3 in the testis, heart, muscle, hepatopancreas and gill. All of the Piwis are found in germ cells of adult testis in P. trituberculatus by in situ hybridization, suggesting that these genes may play function during spermiogenesis in this species. [Copyright &y& Elsevier]