Oxygen radicals regulate many physiological processes, such as signaling, proliferation, and apoptosis, and thus play a pivotal role in pathophysiology and disease development. There are at least two thioredoxin reductase/ thioredoxin/peroxiredoxin systems participating in the cellular defense against oxygen radicals. At present, relatively little is known about the contribution of individual enzymes to the redox metabolism in different cell types. To begin to address this question, we generated and characterized mice lacking functional mitochondrial thioredoxin reductase (TrxR2). Ubiquitous Cre-mediated inactivation of TrxR2 is associated with embryonic death at embryonic day 13. TrxR2 ؊/؊ embryos are smaller and severely anemic and show increased apoptosis in the liver. The size of hematopoietic colonies cultured ex vivo is dramatically reduced. TrxR2-deficient embryonic fibroblasts are highly sensitive to endogenous oxygen radicals when glutathione synthesis is inhibited. Besides the defect in hematopoiesis, the ventricular heart wall of TrxR2 ؊/؊ embryos is thinned and proliferation of cardiomyocytes is decreased. Cardiac tissue-restricted ablation of TrxR2 results in fatal dilated cardiomyopathy, a condition reminiscent of that in Keshan disease and Friedreich's ataxia. We conclude that TrxR2 plays a pivotal role in both hematopoiesis and heart function.Reactive oxygen species (ROS)-generated mainly as a byproduct of the respiratory chain or by oxidases-are implicated in the pathogenesis and pathophysiology of a variety of human diseases such as cancer, cardiovascular, and degenerative disorders. A variety of cellular antioxidant systems control the balance of free intra-and extracellular oxygen radicals. Previous efforts have addressed the physiological role of superoxide dismutases, catalases, and glutathione (GSH) peroxidases in vivo, but the role of the thioredoxin/thioredoxin reductase/ peroxiredoxin system in ROS removal has only recently attracted attention.Thioredoxins are small redox-active proteins with an essential function in DNA metabolism and repair, transcription, and cell-cell communication (1). Acting through peroxiredoxins, they also efficiently protect cells from oxidative damage (27). Cytosolic (Trx1) and mitochondrial (Trx2) thioredoxins are required for proliferation and protection from apoptosis during early embryogenesis (26). Moreover, in chicken B cells, Trx2 is critically involved in the regulation of mitochondriondependent apoptosis (37). More recently, heart-specific overexpression of dominant-negative Trx1 was shown to be associated with increased oxidative stress and cardiac hypertrophy in mice (39).Trx activities are governed by thioredoxin reductases (TrxRs) that, in turn, use NADPH/H ϩ as the reducing agent (23). TrxRs are members of the pyridine nucleotide-disulfide oxidoreductase family, form homodimers, and possess two interacting redox-active centers. The C-terminal redox center contains a catalytically important selenocysteine (SeCys) (9,17,41). In mammals, three TrxRs...

Two distinct thioredoxin/thioredoxin reductase systems are present in the cytosol and the mitochondria of mammalian cells. Thioredoxins (Txn), the main substrates of thioredoxin reductases (Txnrd), are involved in numerous physiological processes, including cell-cell communication, redox metabolism, proliferation, and apoptosis. To investigate the individual contribution of mitochondrial (Txnrd2) and cytoplasmic (Txnrd1) thioredoxin reductases in vivo, we generated a mouse strain with a conditionally targeted deletion of Txnrd1. We show here that the ubiquitous Cre-mediated inactivation of Txnrd1 leads to early embryonic lethality. Homozygous mutant embryos display severe growth retardation and fail to turn. In accordance with the observed growth impairment in vivo, Txnrd1-deficient embryonic fibroblasts do not proliferate in vitro. In contrast, ex vivo-cultured embryonic Txnrd1-deficient cardiomyocytes are not affected, and mice with a heartspecific inactivation of Txnrd1 develop normally and appear healthy. Our results indicate that Txnrd1 plays an essential role during embryogenesis in most developing tissues except the heart. Thioredoxins (Txn) are small redox-reactive proteins that regulate many cellular processes (3). Txn exert a cytokine-like influence on blood cells (29), modulate the activity of redoxregulated transcription factors such as NF-B (34) and AP-1 (20), mediate peroxiredoxin antioxidant properties (35), and are putatively involved in DNA synthesis. The activity of Txn is controlled by thioredoxin reductases (Txnrd) together with NADPH as a cofactor. Three mammalian thioredoxin reductases are known, including a cytosolic (Txnrd1) (10), a mitochondrial (Txnrd2) (9), and a testis-specific (41) isoform. Thioredoxin reductases are homodimeric flavoproteins with two N-and C-terminally located interacting catalytic centers (12,26). The C-terminal redox center contains a selenocysteine (Sec) residue which is part of a conserved Gly-Cys-Sec-Gly motif that is crucial for Txnrd function. Besides thioredoxins, thioredoxin reductases can also reduce other substrates, such as lipoic acid, NK-lysin, ascorbate, and ubiquinone (1, 31, 45).Several gene targeting approaches with mice have been performed to investigate the participation of the Txn/Txnrd systems in development and adult physiology. The results revealed that cytosolic (Txn1) and mitochondrial (Txn2) thioredoxins are indispensable for embryonic development (24, 30). In Txn1 Ϫ/Ϫ mutants, early embryonic death (by embryonic day 6.5 [E6.5]) is associated with a dramatically reduced proliferation of inner mass cells. Txn2-deficient embryos develop exencephaly, show markedly increased apoptosis, and die during midgestation around E10.5. Using a conditional targeting approach, we have shown recently that Txnrd2 is also essential for embryonic development (5). Txnrd2-null embryos die around E13.0 due to defects in hematopoiesis and heart development. The cardiac-specific deletion of Txnrd2 leads to fatal dilated cardiomyopathy and morphological abnormalit...

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.Sperm chromatin condensation during the final steps of spermatogenesis in mammals is a multistep process that includes the sequential replacement of the majority of histones by transition proteins and protamines in testis (6, 7). During epididymal transit of spermatozoa, protamine thiol oxidation is completed and intra-and intermolecular cross-links are formed. Hence, a transcriptionally inactive and tightly packed haploid genome is generated rendering sperm nuclei more resistant to mechanical and chemical insults (2). Recently, Cho and colleagues showed that chimeric mice hemizygous for protamine 1 or 2 fail to transmit the targeted allele to the germ line (8).Selenium depletion studies of rodents clearly demonstrated the importance of this trace element in male fertility. Third generation selenium deficiency is associated with structural abnormalities, such as broken midpieces of sperm tails, giant heads, and reversible testicular atrophy (5, 34). Due to its particular high expression in mammalian testis (21) and its resistance to selenium deprivation in testis, the selenoenzyme phospholipid hydroperoxide glutathione peroxidase (PHGPx) is thought to account for most of the defects associated with severe selenium deficiency.PHGPx was initially characterized as a lipid peroxidationinhibiting protein (33) and was later shown to be an unusual member of the glutathione peroxidase family, in particular for its scarce specificity for both the oxidizing and reducing substrates (32). Most relevant in this respect was the observation that, in the presence of low glutathione (GSH) concentration, specific protein -SH groups may act as a reductant in the catalytic cycle with a stoichiometry of 2 equivalents of thiol per mole of hydroperoxide (13,22,...