Antonella Roveri

Diversity of glutathione peroxidases.

Publisher Summary This chapter focuses on the diversity of glutathione peroxidases. Selenium was identified as a toxic factor for grazing animals in the first half of the twentieth century and since then has been considered hazardous. Only long after the identification of the first selenoenzymes in bacteria and mammals was a Recommended Dietary Allowance gradually established. In fact, the putative biological roles of the selenoenzymes, particularly those of the glutathione peroxidases (GPX), proved instrumental in the understanding of selenium deficiency syndromes in livestock and humans, although the emerging complexity of selenium enzymology still precludes definitive conclusions. The selenium-dependent peroxidases have long been considered a late achievement of evolution, as they were only detected in vertebrates. This view now has to be revised. Whether the common ancester of the GPX superfamily was a selenoprotein or a cysteine-containing homolog cannot be deduced from the available sequences. The only prokaryotic member of the superfamily detected so far, a cobalamine-binding protein of Escherichia coli , does not contain selenocysteine, and despite ongoing efforts, functionally active glutathione peroxidases have not yet been found in prokaryotes.

Male Fertility Is Linked to the Selenoprotein Phospholipid Hydroperoxide Glutathione Peroxidase

Abstract The selenoprotein phospholipid hydroperoxide glutathione peroxidase (PHGPx) accounts for almost the entire selenium content of mammalian testis. PHGPx is abundantly expressed in spermatids as active peroxidase but is transformed to an oxidatively inactivated protein in mature sperm, where it is a major constituent of the mitochondrial capsule in the midpiece. Male infertility in selenium-deficient animals, which is characterized by impaired sperm motility and morphological midpiece alterations, is considered to result from insufficient PHGPx content. We studied the relationship between sperm PHGPx, measured as rescued activity, and human fertility. Sperm specimens from 75 infertile men and 37 controls were analyzed for fertility-related parameters according to World Health Organization criteria. The PHGPx protein content was estimated after reductive solubilization of the spermatozoa by measuring the rescued PHGPx activity. Rescued PHGPx activity of infertile men ranged significantly below that of controls (93.2 ± 60.1 units/mg sperm protein vs. 187.5 ± 55.3 units/mg) and was particularly low in oligoasthenozoospermic specimens (61.93 ± 45.42 units/mg; P < 0.001 compared with controls and asthenozoospermic samples). Rescued PHGPx activity was correlated positively with viability, morphological integrity, and most profoundly forward motility (r = 0.35, 0.44, and 0.45, respectively). In isolated motile samples, motility decreased faster with decreasing PHGPx content. In humans, PHGPx appears to be indispensable for structural integrity of spermatozoa and to codetermine sperm motility and viability. Because the content of PHGPx, irrespective of the cause of alteration, is correlated with fertility-related parameters, PHGPx can be considered a predictive measure for fertilization capacity.

Mitochondrial glutathione peroxidase 4 disruption causes male infertility

Selenium is linked to male fertility. Glutathione peroxidase 4 (GPx4), first described as an antioxidant enzyme, is the predominant selenoenzyme in testis and has been suspected of being vital for spermatogenesis. Cytosolic, mitochondrial, and nuclear isoforms are all encoded by the same gene. While disruption of entire GPx4 causes early embryonic lethality in mice, inactivation of nuclear GPx4 does not impair embryonic development or fertility. Here, we show that deletion of mitochondrial GPx4 (mGPx4) allows both normal embryogenesis and postnatal development, but causes male infertility. Infertility was associated with impaired sperm quality and severe structural abnormalities in the midpiece of spermatozoa. Knockout sperm display higher protein thiol content and recapitulate features typical of severe selenodeficiency. Interestingly, male infertility induced by mGPx4 depletion could be bypassed by intracytoplasmic sperm injection. We also show for the first time that mGPx4 is the prevailing GPx4 product in male germ cells and that mGPx4 disruption has no effect on proliferation or apoptosis of germinal or somatic tissue. Our study finally establishes that mitochondrial GPx4 confers the vital role of selenium in mammalian male fertility and identifies cytosolic GPx4 as the only GPx4 isoform being essential for embryonic development and apoptosis regulation.—Schneider, M., Forster, H., Boersma, A., Seiler, A., Wehnes, H., Sinowatz, F., Neumüller, C., Deutsch, M. J., Walch, A., Hrabede Angelis, M., Wurst, W., Ursini, F., Roveri, A., Maleszewski, M., Maiorino, M. Conrad, M. Mitochondrial glutathione peroxidase 4 disruption causes male infertility. FASEB J. 23, 3233–3242 (2009). www.fasebj.org

The Nuclear Form of Phospholipid Hydroperoxide Glutathione Peroxidase Is a Protein Thiol Peroxidase Contributing to Sperm Chromatin Stability

ABSTRACT The selenoenzyme phospholipid hydroperoxide glutathione peroxidase (PHGPx) is regarded as the major molecular target of selenodeficiency in rodents, accounting for most of the histopathological and structural abnormalities of testicular tissue and male germ cells. PHGPx exists as a cytosolic form, mitochondrial form, and nuclear form (nPHGPx) predominantly expressed in late spermatids and spermatozoa. Here, we demonstrate that mice with a targeted deletion of the nPHGPx gene were, unlike mice with the full knockout (KO) of PHGPx, not only viable but also, surprisingly, fully fertile. While both morphological analysis of testis and epididymis and sperm parameter measurements did not show any apparent abnormality, toluidine blue and acridine orange stainings of spermatozoa indicated defective chromatin condensation in the KO sperm isolated from the caput epididymis. Furthermore, upon drying and hydrating, KO sperm exhibited a significant proportion of morphologically abnormal heads. Monobromobimane labeling and protein-free thiol titration revealed significantly less extensive oxidation in the cauda epididymis when compared to that in the wild type. We conclude that nPHGPx, by acting as a protein thiol peroxidase in vivo, contributes to the structural stability of sperm chromatin.

Enzymatic and immunological measurements of soluble and membrane-bound phospholipid-hydroperoxide glutathione peroxidase

Publisher Summary Phospholipid-hydroperoxide glutathione peroxidase (PHGPx) is a selenoenzyme in which selenium is present in the active site as selenocysteine. PHGPx reduces phospholipid hydroperoxides and small soluble hydroperoxides. PHGPx is also competent for the reduction of cholesterol and cholesterol ester hydroperoxides in liposomes, membranes, and oxidized low-density lipoproteins. This capability to react with different hydroperoxides accounts for the evidence that all titrable hydroperoxide groups generated in microsomal membranes during lipid peroxidation are reduced by this enzyme, while being resistant to classic glutathione peroxidase (GPx). The observation that microsomal lipid peroxidation is inhibited by PHGPx and glutathione only if the membranes contain a physiological amount of vitamin E, suggests a tandem mechanism for the two antioxidant activities. PHGPx, therefore, prevents the formation of new peroxidation chains and spares vitamin E. Membrane-bound PHGPx from testes shows the same substrate specificity, electrophoretic mobility, immunological reactivity, and peptide mapping with cyanogen bromide and SV-8 protease with respect to the cytosolic form.

Microsomal lipid peroxidation: effect of vitamin E and its functional interaction with phospholipid hydroperoxide glutathione peroxidase.

The role of vitamin E in the protection against iron dependent lipid peroxidation was studied in rat liver microsomes and Triton-dispersed microsomal lipid micelles. In these systems, an antioxidant effect of vitamin E at a physiological ratio to phospholipids could be observed only in the presence of phospholipid hydroperoxide glutathione peroxidase (PHGPX) and glutathione. The rationale of this cooperation is discussed on the basis of the hydroperoxyl radical scavenging capacity of vitamin E and the reduction of membrane hydroperoxides by PHGPX. The scavenging of lipid hydroperoxyl radicals by vitamin E, although inhibiting propagation of the peroxidative chain, produces lipid hydroperoxides from which ferrous iron generates alkoxyl radicals that react with vitamin E almost as fast as with fatty acids. Therefore, only if membrane hydroperoxides are continuously reduced by this specific peroxidase does the scavenging of hydroperoxyl radicals by vitamin E lead to an effective inhibition of lipid peroxidation.

Kinetic mechanism and substrate specificity of glutathione peroxidase activity of ebselen (PZ51)

The glutathione peroxidase activity of ebselen (PZ51) was studied using different hydroperoxidic substrates. The single progression curves obtained in the spectrophotometric test were processed by a computer to fit the integrated rate equation that describes the ping pong reaction of the Se glutathione peroxidase. Ebselen catalyzes the GSH peroxidase reaction with a mechanism that appears kinetically identical to the mechanism of the enzymes. The inactivation of the catalytic properties of ebselen by iodoacetate suggests that a selenol moiety is involved. Among the substrates tested, the best hydroperoxidic substrates are the hydroperoxy derivatives of phosphatidyl choline. Ebselen is active also on membrane hydroperoxides as does phospholipid hydroperoxide glutathione peroxidase but not glutathione peroxidase.

Phospholipid Hydroperoxide Glutathione Peroxidase is a Seleno-Enzyme Distinct from the Classical Glutathione Peroxidase as Evident from Cdna and Amino Acid Sequencing

The primary structure of phospholipid hydroperoxide glutathione peroxidase (PHGPx) was partially elucidated by sequencing peptides obtained by cyanogen bromide cleavage and tryptic digestion and by isolating and sequencing corresponding cDNA fragments covering about 75% of the total sequence. Based on these data PHGPx can be rated as a selenoprotein homologous, but poorly related to classical glutathione peroxidase (GPx). Peptide loops constituting the active site in GPx are, however, strongly conserved in PHGPx. This suggests that the mechanism of action involving an oxidation/reduction cycle of a selenocysteine residue is essentially identical in PHGPx and GPx.

Testosterone mediates expression of the selenoprotein PHGPx by induction of spermatogenesis and not by direct transcriptional gene activation

Selenium deficiency is known to be associated with male infertility, and the selenoprotein PHGPx has been shown to increase in rat testis after puberty and to depend on gonadotropin stimulation in hypophysectomized rats [Roveri et al. (1992) J. Biol. Chem. 267, 6142–6146]. Exposure of decapsulated whole testis, however, failed to reveal any transcriptional activation or inhibition of the PHGPx gene by testosterone, human chorionic gonadotropin, or forskolin. Nevertheless, it was verified that the specific activity of PHGPx in testis, but not of cGPx, correlated with sexual maturation. Leydig cell destruction in vivo by ethane dimethane sulfonate (EDS) resulted in a delayed decrease in PHGPx activity and mRNA that could be completely prevented by testosterone substitution. cGPx transiently increased upon EDS treatment, probably as a result of reactive macrophage augmentation. In situ mRNA hybridization studies demonstrated an uncharacteristic low level of cGPx transcription in testis, whereas PHGPx mRNA was abundantly and preferentially expressed in round spermatids. The data show that the age or gonadotropin‐dependent expression of PHGPx in testis does not result from direct transcriptional gene activation by testosterone, but is due to differentiation stage‐specific expression in late spermatids, which are under the control of Leydig cell‐derived testosterone. The striking burst of PHGPx expression at the transition of round to elongated spermatids suggests an involvement of this selenoprotein in sperm maturation.—Maiorino, M., Wissing, J. B., Brigelius‐ Flohe´, R., Calabrese, F., Roveri, A., Steinert, P., Ursini, F., Flohe´, L. Testosterone mediates expression of the selenoprotein PHGPx by induction of spermatogenesis and not by direct transcriptional gene activation. FASEB J. 12, 1359–1370 (1998)

Phospholipid hydroperoxide glutathione peroxidase: specific activity in tissues of rats of different age and comparison with other glutathione peroxidases

The tissue distribution of phospholipid hydroperoxide glutathione peroxidase (PHGPX) was studied in rats of different ages. In the same samples the activities of Se-dependent glutathione peroxidase (GPX), and non-Se-dependent glutathione peroxidase (non Se-GPX) were also determined using specific substrates for each enzyme. Enzymatically generated phospholipid hydroperoxides were used as substrate for PHGPX, hydrogen peroxide for GPX, and cumene hydroperoxide for non-Se-GPX (after correction for the activity of GPX on this substrate). PHGPX specific activity in different organs is as follows: liver = kidney greater than heart = lung = brain greater than muscle. Furthermore, this activity is reasonably constant in different age groups, with a lower specific activity observed only in kidney and liver of young animals. GPX activity is expressed as follows: liver greater than kidney greater than heart greater than lung greater than brain = muscle, and substantial age-dependent differences have been observed (adult greater than old greater than young). Non-Se-GPX activity was present in significant amount only in liver greater than lung greater than heart and only in adult animals. These results suggest a tissue- and age-specific expression of different peroxidases.