Glutathione S-transferases (GSTs) of helminths are multifunctional enzymes that neutralize endo- and exogenous bio-reactive free radicals and chemical compounds by catalyzing conjugation of reduced glutathione. GSTs bind non-substrate ligands in cleft formed in the center of subunit dimer and increase their solubility and transfer for excretion (Sluis-Cremer et al., 1996). Furthermore, nematode GSTs have been suggested to suppress or subvert host immunity, because of its prostaglandin biosynthesis activity (Meyer et al., 1996).

GSTs are classified into families named as alpha, mu, pi, sigma, and theta, according to the substrate specificity, immunogenic reactivity, and amino acid sequence of their polypeptides (Brophy and Pritchard, 1994; Hayes and Pulford, 1995). Biochemical features of helminth GSTs appeared like mosaics of family characters of mammalian GSTs, therefore, it was proposed that helminth GSTs should be classified into three superfamilies, independent of mammalian GST families (Brophy and Pritchard, 1994).

Of the helminth GSTs, 28 kDa GSTs of trematodes (28GSTs) are localized in tegument, parenchyma and genital organs (Taylor et al., 1988; Liu et al., 1996; Gobert et al., 1998). From its enzymatic activity and tissue localization, 28GST has been shown to play a major role in phase II detoxification system for the fluke's survival (Brophy and Pritchard, 1994). 28GST cDNA was cloned from Clonorchis sinensis, and the recombinant protein was produced. Recombinant C. sinensis 28GST had enzymatic activity and was grouped into class sigma. Immunohistochemical staining revealed localization of 28GST in tegument, parenchyma and intra-uterine eggs of adult C. sinensis (Kang et al., 2001). This study was undertaken to further elucidate fine distribution and possible functions of 28GST in adult C. sinensis.

Clonorchis sinensis metacercariae were collected from topmouth gudgeons, Pseudorasbora parva, by artificial digestion. Adult C. sinensis were recovered from bile ducts of experimental rabbits 7 months after infection with the metacercariae. Flukes were fixed in a solution of 2% paraformaldehyde/0.4% glutaraldehyde, pH 7.4 for an hour at room temperature, and they were dehydrated in a series of alcohol, and embedded in LR gold resin (London Resin Co., UK) and polymerized under UV light. Ultrathin ribbons were cut at 90 nm thickness and mounted on nickel grids. The ultrathin ribbons were immunogold-labeled by the procedure described previously (Yu and Chai, 1995). Mouse monoclonal antibody (CTHK-17) raised against recombinant Cs28GST was used as primary antibody (Kang et al., 2001), and goat anti-mouse IgG, 5 nm colloidal gold-conjugated (Sigma), at 1:70 dilution was used as secondary antibody. Stained ribbons were observed under a transmission electron microscope (Jeol 1200EXII, Tokyo, Japan).

Tegumental syncytium was moderately labeled with gold particles (Fig. 1), and in tegumental syncytium gold particles were associated with electron dense granules and increased distally toward cytoplasmic membrane. Cytons were irregular shapes and distinguished from subtegumental cells by darker stainability of cytoplasm. Cytons were labeled densely with gold particles (Fig. 2), and there appeared a tendency that gold particles were more dense and associated with electron dense granules in the periphery of cytons. Mitochondria were abundant around nucleus. Tails of sperms in vas efferens near ovary were labeled heavily with gold particles but sperm body was not (Fig. 3). Low density of gold particle was found in parenchymal tissues.

In adult C. sinensis, 28GST was abundantly distributed in subtegumental parenchymal tissues and less extent in tegumental syncytium (Kang et al. 2001). Ultrastructurally, moderate level of 28GST was present in cytons and low level in syncytium. Distribution pattern of C. sinensis 28GST was similar to that of schistosomes (Taylor et al., 1988; Liu et al., 1996; Gobert et al., 1998). It is known that 28GST is produced in cyton and transferred to tegumental syncytium through cytoplasmic processes. In Schistosoma japonicum, 28GST is present as a major protein on body surface (Henkle et al., 1990). Molecules associated with tegument are secreted or released when cytoplasmic membrane is turned over (McLaren, 1980), and presented to host as stimulatory/reactive bio-compounds. GSTs are one of the anti-oxidant molecules distributed in host-parasite interface, tegument and subtegumental parenchymal tissues (Mei and LoVerde, 1997; Hong et al., 2001), and 28GSTs neutralize exogenous bio-reactive compounds as well as endogenous ones (Kang et al., 2001) and play a key role in defense physiology of trematodes.

In the present study, 28GST was shown to be localized most densely in sperm tails of of C. sinensis. Isoform of class Mu GST is present in human testes (Hussey and Hayes, 1993) and abundant in fluid of seminiferous tubules of rat testes (Mukherjee et al., 1999). Sertoli cells are prime secretors of Mu GSTs in rat testes. 28GST was also shown to be present in male reproductive organs of S. mansoni (Liu et al., 1996). Testosterone binds to 28GST of Schistosoma haematobium and inhibits its enzymatic activity in vitro (Remoue et al., 2002). Sex-dependent immune response to S. haematobium 28GST was revealed in infected adult human subjects. These findings imply that 28GST play a key role in metabolism and physiology of reproductive system of flukes. Challenge infection of experimental animals immunized with schistosome GSTs results in decrease of worm fecundity (Boulanger et al., 1991). In the course of egg formation, fertilization and embedding of oocytes inside the eggs increased expression of 28GST (Liu et al., 1996). Intra-uterine eggs of C. sinensis contained large amount of 28GST (Kang et al., 2001). In life history of C. sinensis, embryos develop to miracidia in intra-uterine eggs. From the above description, it, therefore, seems that abundancy of 28GST in the eggs may be related to the proliferation of embryonic cells. In conclusion, it is highly possible that 28GST could play a key role in fecundity of C. sinensis flukes.