Epididymal specific, selenium-independent GPX5 protects cells from oxidative stress-induced lipid peroxidation and DNA mutation

Abstract

study qustion: Can selenium (Se) independent, epididymal-specific glutathione peroxidase 5 (GPX5) protect CHO-K1 cells from oxidative damage and, more specifically, from lipid peroxidation and DNA mutation?summary answer: CHO-K1 cells expressing GPX5 have increased resistance to oxidative challenge and, more specifically, decreased levels of lipid peroxidation and decreased levels of the downstream DNA lesion 8-oxo-7,8-dihydroguanine (8-oxodG) what is known already: GPX5 associates with sperm during transit of the epididymis, and has been postulated to protect sperm from peroxide-mediated attack. However, its function as an active glutathione peroxidase has been questioned due to substitution of the classical selenocysteine residue at its active site. Indirect evidence for a functional role for GPX5 has been provided by in vivo studies, in particular from the GPX5 knockout mouse whereby offspring sired by GPX5 2/2 males have a higher rate of spontaneous abortion and developmental defects, attributed to increased oxidative injury (8-oxodG) to sperm DNA, but only when the GPX5 2/2 males are over 1 year of age. Interestingly, we have previously shown severely reduced levels of GPX5 in humans.study design, size, duration: To look more directly at its role in protection against oxidative damage, we have used an in vitro system, generating a CHO-K1 mammalian cell line expressing recombinant rat GPX5.participants/materials, setting, methods: We have used the recombinant CHO-K1 cells to determine whether GPX5 is able to protect these cells from an administered oxidative challenge, using a range of approaches. We compared the viability of GPX5-expressing cells with control cells by both MTT and trypan blue exclusion assays. We next investigated whether GPX5 protects the cells specifically from lipid peroxidation, by using the fluorescent reporter molecule C11-BODIPY (581/591) , and thus from downstream DNA mutation, by comparing levels of the DNA lesion 8-oxodG. We also investigated whether GPX5 can be transferred to rat sperm via epididymosomes.main results and the role of chance: GPX5-expressing CHO-K1 cells had increased viability compared with control cells following oxidative challenge (P , 0.005). We also found that GPX5-expressing CHO-K1 cells had significantly lower levels of C11-BODIPY (581/591) oxidation, and hence lipid peroxidation, compared with control cells. Levels of 8-oxodG DNA damage were also markedly lower in the nuclei of GPX5-expressing cells than in control cells. Finally, we showed that GPX5 can be transferred to rat sperm via epididymosomes.limitations, reasons for caution: GPX5 is not active in glutathione peroxidase assays using H 2 O 2 as the substrate. However, the related non-mammalian Se-independent GPXs show preference for electron donors other than glutathione, with a number utilizing thioredoxin as a reducing equivalent. Hence, the in vitro activity of GPX5 needs to be assessed using a range of alternative substrates and electron donors. GPX5 is secreted by the epididymis and associate...

In vivo oxidative stress alters thiol redox status of peroxiredoxin 1 and 6 and impairs rat sperm quality

“…Since SOD1 quantities did not change due to the tert-BHP treatment, we can conclude that the reabsorption of residual cytoplasm is little or not compromised and thus an active transfer of PRDXs from the epididymal epithelium to the maturing spermatozoa may occur. Although we did not probe a direct transfer of PRDXs to spermatozoa from treated rats, previous evidence strongly suggest this possibility; epididymosomes from bull and humans contain PRDXs3233 and others already demonstrated that proteins can be transferred from epididymosomes to spermatozoa 34353637…”

“…It is also possible that other antioxidant enzymes may be transferred by epididymosomes, along with PRDXs; these organelles also carry SOD1, thioredoxins, glutathione peroxidase 5 and s-glutathione transferases 3233343537. However, this transfer seems to be well regulated since SOD1 amounts were not augmented in spermatozoa from treated rats.…”

New insights into epididymal function in relation to sperm maturation

“…The most realistic function is protection of the sperm during epididymal transit. Several proteins successively secreted in the epididymal fluid may be involved in the reduction of reactive oxygen species in the luminal fluid such as GPX5 (Chabory et al 2010, Taylor et al 2013, thioredoxin, GSTM1-3, SOD1, and PRDX2-5. This protective function may also be illustrated by the presence of several proteins and peptides such as several b-defensins (Yamaguchi & Ouchi 2012), lipocalins, and CRES proteins against bacterial attack .…”

Antioxidant enzyme expression of mRNA and protein in the epididymis of finasteride-treated male rat offspring during postnatal development

“…In light of this danger caused by SODs, the presence of the epididymal secretory form of glutathione peroxidase (GPX5) which covers the spermatozoa head is noticeable [16,28]. It has been demonstrated that GPX5 protects cells from oxidative stress induced lipid peroxidation and DNA mutation [67], maintains sperm DNA integrity [68] and regulates the synthesis of prostaglandin, key signaling molecules needed by spermatozoa [69]. Therefore, the decrease of GPX5 in the adult F1:Fin rats observed in our study could result in inadequate spermatozoa protection and subsequent dysfunction.…”