Molecular and histological evaluation of tributyltin toxicity on spermatogenesis in a marine fish, the mummichog ( Fundulus heteroclitus)

There is still concern about the effects of organotin compounds (OTs) on marine organisms, and especially on their reproductive systems. We investigated the toxicity of tributyltin oxide (TBTO) on spermatogenesis in a marine fish, mummichog, Fundulus heteroclitus. TBTO exposure caused serious histological damage to the testis, including reduction in counts of spermatids and spermatozoa and malformation of somatic cells around the seminal duct. Analysis of the incorporation of bromodeoxyuridine into spermatogenic cells revealed inhibition of the proliferation of germ cells. To find a biomarker for evaluation of the effects of TBTO on fish spermatogenesis, we cloned genes downregulated by TBTO exposure in the mummichog testis, and identified mummichog creatine kinase (mCK). The cDNA sequence of mCK contained an open reading frame encoding 387 amino acid residues ( M r = 43,344). The derived amino acid sequence of mCK was very similar to that of the testicular-type CK of the rainbow trout, Oncorhynchus mykiss. Furthermore, Northern blot analysis revealed that mCK was produced specifically in the testis. We therefore identified mCK in the mummichog as a testicular-type CK. Real-time PCR revealed that exposure of the fish to TBTO significantly reduced mCK expression in the testis. To some extent, this reduction was coincident with that of bromodeoxyuridine incorporation into spermatogenic cells. The mCK gene can therefore be used as a biomarker for evaluating the effects of TBTO on fish spermatogenesis. In addition, levels of expression of the mCK gene in control fish were well correlated with increments in the gonad somatic index (GSI) below 4%. Individuals that were thought to have testicular damage caused by TBTO could be discriminated from those considered normal. The results suggest that TBTO is involved in the suppression of fish spermatogenesis and that analysis of both GSI values and mCK gene expression is useful for evaluating the levels of xenobiotic pollution in coastal areas.