Novel functional association of rat testicular membrane-associated cytosolic glutathione S transferases and cyclooxygenase in vitro.

AIM: To analyze the role of cytosolic glutathione S-transferases cGSTs and membrane-associated cytosolic GSTs macGSTs in prostaglandin biosynthesis and to evaluate the possible interaction between glutathione S-transferases GSTs and cyclooxygenase (COX) in vitro. METHODS: SDS-PAGE analysis was undertaken for characterization of GSTs, thin layer chromatography (TLC) to monitor the effect of GSTs on prostaglandin biosynthesis from arachidonic acid (AA) and spectrophotometric assays were done for measuring activity levels of COX and GSTs. RESULTS: SDS-PAGE analysis indicates that macGSTs have molecular weights in the range of 25-28 kDa. In a coupled assay involving GSTs, arachidonic acid and cyclooxygenase-1, rat testicular macGSTs produced prostaglandin E2 and F2alfa, while the cGSTs caused the generation of prostaglandin D2, E2 and F2alfa. In vitro interaction studies on GSTs and COX at the protein level have shown dose-dependent inhibition of COX activity by macGSTs and vice versa. This effect, however, is not seen with cGSTs. The inhibitory effect of COX on macGST activity was relieved with increasing concentrations of reduced glutathione (GSH) but not with 1-chloro 2,4-dinitrobenzene (CDNB). The inhibition of COX by macGSTs, on the other hand, was potentiated by glutathione. CONCLUSION: We isolated and purified macGSTs and cGSTs from rat testis and analyzed their involvement in prostaglandin biosynthesis. These studies reveal a reversible functional interaction between macGSTs and COX in vitro, with possible interactions between them at the GSH binding site of macGSTs.

Expression of cyclooxygenase-2 in rat testis.

Cyclooxygenase, the rate-limiting enzyme in the production of prostaglandins, exists in two isoforms, the constitutive cyclooxygenase 1 (Cox-1) and the inducible cyclooxygenase 2 (Cox-2). Cox-1 is involved in homeostatic functions while Cox-2 is implicated in various pathological processes such as inflammation and cancer. The present study describes the constitutive expression of Cox-2 in immature and mature rat testis, primarily localized in the spermatogonial cells. An interesting observation is the presence of Cox-2 on the chromatin and also in cytosol, apart from nuclear and endoplasmic reticular membranes. The significance of this observation is not yet clear, though its presence in the nucleus raises the possibility of Cox-2 having a more direct role in gene regulation than was thought earlier. In addition, the Cox-2 mRNA in testis is the smaller (2.8 kb) of the two isoform transcripts reported for Cox-2. Further hormone treatment regimes (testosterone/follicle stimulating hormone) increased the levels of Cox-2 protein within 6 h after treatment, suggesting that the sustained levels of Cox-2 protein in testis can be further influenced by gonadotrophins and androgens.

Purification and characterization of rat testicular glutathione S-transferases: role in the synthesis of eicosanoids.

AIM: Purification of glutathione S-transferases (GSTs) from rat testis; separation and identification of various subunits and their role in eicosanoid biosynthesis. METHODS: Purification of rat testicular GSTs by affinity chromatography, employing S-hexylglutathione-linked epoxy-activated Sepharose 6B column and separation of individual subunits by reverse phase-high pressure liquid chromatography (RP-HPLC). Characterization of affinity purified GSTs by Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. The role of testicular GSTs in eicosanoid biosynthesis was determine by incubating GSTs with 5, 6-Leukotriene A4Me (LTA4Me) and prostaglandin H2(PGH2) and analyzing the products formed on HPLC/TLC. RESULTS: The present study reveals that majority of rat testicular GSTs are of Yb size (60%) with molecular weight of 27 kDa. The most predominant subunits, however, are GST Yn2(27%), followed by GST Yc(24%) and GST Yn1(20%). These testicular GSTs showed very high Leukotriene C4 (LTC4) synthase activity with 5, 6-Leukotriene A4Me (LTA4Me) as the substrate and prostaglandin D (PGD) synthase activity with prostaglandin H2(PGH2) as the substrate. CONCLUSION: Majority of rat testicular GSTs are Yb sized and are involved in the synthesis of eicosanoids like LTC4 and PGD2.