Isolation and identification of metallothionein isoforms (MT-1 and MT-2) in the rat testis

Abstract

It has been a long-lasting controversial issue as to whether or not the male genital organs, such as the testis and prostate, contain metallothioneins (MTs), a group of cysteine-rich heavy-metal-binding proteins that play a role in detoxifying heavy metals such as cadmium (Cd). Earlier studies reported that the rodent testis lacks MTs and concluded that this is why the testis is very susceptible to Cd, although other indirect experimental evidence suggests that MTs are present in this organ. A deficiency of MTs in the testis was originally suspected on the basis of amino acid composition analysis, since MT-like proteins isolated as Cd-binding proteins did not have a characteristic MT structure. In the present study, we demonstrate that the rat testis indeed expresses Cd-binding proteins with sequences identical to those previously described for MT-1 and MT-2, the major isoforms. To confirm that MT-1 and MT-2 are present in the rat testis, we purified and isolated Cd-binding proteins by homogenization using Cd-containing buffer, followed by sequential purification using Sephadex G-75 gel filtration chromatography and anion HPLC column chromatography, which yielded Cd-binding protein-1 (Cd-BP-1) and -2 (Cd-BP-2). After pyridylethylation, Cd-BP-1 and Cd-BP-2 were subjected to specific protein fragmentation by acids and endopeptidases, which revealed that these Cd-binding proteins have the same primary structures as MT-1 and MT-2 respectively. Thus we believe that the present results clearly resolve the long-standing debate about the presence of MTs in the testis, at least in the rodent.

Metallothioneins and diseases with special reference to cadmium poisoning

“…These data are in accordance with data published by Suzuki et al [37]. The long-standing debate about whether MT is expressed in testicular tissue thus seems to be resolved.…”

Tissue-Dependent Induction of Heme Oxygenase-1 and Metallothionein-1/2 by Methyl Methanesulfonate

“…In the testis, a blood-testicular barrier has been formed as a defense mechanism against the approach of various xenobiotic substances. Moreover, oxidative-stress responsive proteins HO-1 and MT-1/2 also express in rat testes (Ozawa et al, 2002;Suzuki et al, 1998). Roles of HO-1 and MT-1/2 in the testes remain to be determined.…”

“…In this study, MT-1/2 was found to express highly in control rat testes, and was not induced by MMS administration. Earlier studies have reported that the rodent testis lacks MT-1/2 (Whanger et al, 1980), indicating that this lack of MT-1/2 is the reason why the testes are very susceptible to Cd, although other experimental evidence suggested that there were mRNAs which hybridized with mouse MT-1 cDNA in rat testes under physiological conditions (Wahba et al, 1994;Suzuki et al, 1998). More recently, evidence has appeared that MT-1/2 exists in rat testes by employing RT-PCR with MT-1 and MT-2 specific primers, and there are also MT-1 and MT-2 proteins (Suzuki et al, 1998).…”

“…Earlier studies have reported that the rodent testis lacks MT-1/2 (Whanger et al, 1980), indicating that this lack of MT-1/2 is the reason why the testes are very susceptible to Cd, although other experimental evidence suggested that there were mRNAs which hybridized with mouse MT-1 cDNA in rat testes under physiological conditions (Wahba et al, 1994;Suzuki et al, 1998). More recently, evidence has appeared that MT-1/2 exists in rat testes by employing RT-PCR with MT-1 and MT-2 specific primers, and there are also MT-1 and MT-2 proteins (Suzuki et al, 1998). However, if that were the case, the question again arises as to why the testes are highly susceptible to Cd insult under physiological conditions, and why MT-1/2 is not inducible in the testes by heavy metals and other stimulatory agents such as lipopolysaccharide (LPS) (De et al, 1991).…”

Ultrastructural Demonstration of Mercury Granules in the Placenta of Metallothionein-Null Pregnant Mice after Exposure to Mercury Vapor

“…The 186b RNA sense and antisense probes for the MT-III from a pPCR-MMT3S plasmid, containing the MT-III coding and noncoding sequences 2021-2206 of the MT-III DNA, were labeled with digoxigenin-UTP (Boehringer Mannheim Biochemica, Germany) (Suzuki et al, 1998). The paraffin sections (5 µm) were mounted on 3 -aminopropyltriethoxysilane-coated slides, dried at 60…”