Characterization And Expression Analysis Of Metallothionein In The Testis Of A Marine Crab,Charybdis Japonica,Exposed To Cadmium

Metallothionein (MT) participates in metallic homeostasis and detoxification in living animals. Lots of researches mainly focused on the roles MT played in development of vertebrates, but its functions during spermiogenesis in Crustacea were unclear.The paper focuses on investigating the contribution of MT to spermiogenesis of Japanese stone crab (Chcrybdis japonica). Spermatogenesis is a highly differentiated and complex process which spermatogonium develops into spermatozoon. Spermatogenesis consists of mitosis, meiosis and spermiogenesis. During spermiogenesis, spermatid is transformed into spermatozoon. The sperm of Japanese stone crab Charybdis japonica (Crustacea:Decapoda:Portunidae) is of aflagellated type. The sperm has a spherical acrosome surrounded by the cup-shaped nucleus, which is distinguished from the brachyuran. We identified Charybdis japonica MT complete cDNA sequence from the total RNA of the testis using degenerated PCR and rapid amplification of cDNA end (RACE). The 587bp MT cDNA contained an 80bp 5’untranslated region, a 333bp 3’untranslated region and a 174bp open reading frame. The MT had 57 amino acids including 19 cysteines. MT containsβN andβc. The protein alignment between MT sequences of Charybdis japonica and other crabs showed high similarities and strong identity in cysteine residues that were vital for MT metal-binding affinity. The tissue expression suggested that MT mRNA is expressed in the testis, hepatopancreas, vesicula seminalis, heart and muscle. In situ hybridization result showed that at the early spermatids, abundant MT mRNA concentrates into proacrosomal vesicle, some in cytoplasm. At the middle spermatids, MT mRNA predominates in the proacrosomal vesicle, some in the membrane complex. At the late spermatids and spermatozoa, MT mRNA localizes in the acrosomal tubule and the narrow band between cup-like nucleus and acrosome. Histochemistry analysis of testis showed that the structure of testis is seriously damaged exposed to cadmium (Cd). Real time PCR indicated that transcriptional activation of MT in testis exposed to Cd is regulated in a dose-and/or time-dependent manner.Our study demonstrates that MT is involved in spermiogenesis and plays important roles in the protection of testis against heavy metal toxicity as it does in mammals. Besides, a sensitive response of testis MT to Cd demonstrates MT is an excellent biomarker candidate for evaluating Cd pollution. It is the first time to reveal the role of MT in crustacean spermiogenesis especially exposed to Cd pollution.