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Our previous study demonstrated that preconditioning by 3-times repetitive mild hypoxia significantly augmented expression of mitochondrial thioredoxin-2 (Trx-2) at 3 h after subsequent acute severe hypoxia in rat hippocampus. However, it was unclear whether this augmentation was due to build up of Trx-2 by mild hypoxia before severe hypoxia or by modification of reaction to severe hypoxia itself. To answer on this question we study the expression level during and after preconditioning without subsequent severe hypoxia. Trx-2 expression was studied by immunocytochemistry 3 h and 24 h after first session and 3 h and 24 h after last session of 3-times (spaced at 24 h) mild hypobaric hypoxia (360 Torr, 2h). At 3 h after 1-time hypoxia (first session of 3-time hypoxia) the total number of Trx-2-immunoreactive cells (Nt) was significantly decreased in contrast with control in CA2, CA3 and DG. The number of cells with intensive expression of Trx-2 (Ni) was reduced in CA1 and CA3. At 24 h after the same 1-time hypoxia Nt was lower than in control and at 3 h time-point in all hippocampal areas studied (CA1, CA2, CA3 and DG); Ni was decreased only compared to control in CA1 and CA3. At 3 h after last session of 3-times hypoxia Nt and Ni were significantly down regulated in comparison with control only in CA1. At 24 h after it Nt was significantly decreased compared to control in CA1, CA2 and CA3 (in DG the decrease was not statistically significant) but in all areas was higher than at 24 h after 1-time hypoxia. Dynamics of Nt changes from 3-hours after single to 24-hours after triple moderate hypoxia had the wave phase character. These findings indicate that Trx-2 expression in most areas of hippocampus was decreased to 24 h after 3-time mild hypoxia. Thus the augmentation of Trx-2 expression in hippocampal neurons of preconditioned animals in response to subsequent severe hypoxia is caused obviously not by Trx-2 accumulation during preconditioning sessions but by modification of reaction to severe impact.

The thioredoxin system, composed of thioredoxin (Trx) and thioredoxin reductase (TrxR), emerges as one of the most important thiol-based systems involved in the maintenance of the cellular redox balance. Thioredoxin-like-1 (TXL-1) is a highly conserved protein comprising an N-terminal Trx domain and a C-terminal domain of unknown function. Here we show that TXL-1 is a substrate for the cytosolic selenoprotein TrxR-1. In situ hybridization experiments demonstrates high expression of Txl-1 mRNA in various areas of central nervous system and also in some reproductive organs. Glucose deprivation, but not hydrogen peroxide treatment, reduced the levels of endogenous TXL-1 protein in HEK-293 cell line. Conversely, overexpression of TXL-1 protects against glucose deprivation-induced cytotoxicity. Taken together, the finding that Txl-1 mRNA is highly expressed in tissues which use glucose as a primary energy source and the modulation of TXL-1 levels upon glucose deprivation indicate that TXL-1 might be involved in the cellular response to sugar starvation stress.

The impact of severe hypoxia and preconditioning on the expression of the mitochondrial antioxidant thioredoxin-2 (Trx-2) in rat hippocampus (CA1, CA2, CA3 fields, and dentate gyrus) and neocortex was studied by immunocytochemistry. The preconditioning consisted of three trials of mild hypobaric hypoxia (360 Torr, 2 hr) spaced at 24 hr. The last trial was followed by severe hypobaric hypoxia (180 Torr, 3 hr) 24 hr later. Both in hippocampus and in neocortex, severe hypobaric hypoxia resulted in enhanced Trx-2 expression at 3 hr, followed by a slight decline in Trx-2 levels, which nevertheless remained increased at 24 hr elsewhere except for the CA1 region. The preconditioning considerably augmented severe hypoxia-induced Trx-2 immunoreactivity, affecting both the number of immunoreactive cells and the intensity of immunostaining. The findings suggest a role for Trx-2 in the formation of brain hypoxic/ischemic tolerance accomplished by the preconditioning.

Induction of endogenous antioxidants is one of the key molecular mechanisms of cell resistance to hypoxia/ischemia. The effect of severe hypoxia on the expression of cytosolic antioxidant thioredoxin-1 (Trx) in hippocampus and neocortex was studied in preconditioned and non-preconditioned rats. The preconditioning consisted of three trials of mild hypobaric hypoxia (360 Torr, 2 h) spaced at 24 h. Twenty-four hours after the last trial rats were subjected to severe hypobaric hypoxia (180 Torr, 3 h). Trx expression was studied by immunocytochemistry. In hippocampus severe hypobaric hypoxia rapidly induced Trx expression, which remained elevated still at 24 h. In neocortex the enhanced expression appeared only at 24 h. The preconditioning significantly augmented severe hypoxia-induced Trx-immunoreactivity at 3 h but not at 24 h. These findings point out that Trx contributes to mechanisms of brain tolerance to hypobaric hypoxia, especially in early periods after the exposure.

Mammalian germ cells are endowed with a complete set of thioredoxins (Trx), a class of redox proteins located in specific structures of the spermatid and sperm tail. We report here the characterization, under normal and pathological conditions, of a novel thioredoxin with a germ line-restricted expression pattern, named spermatocyte/spermatid-specific thioredoxin-3 (SPTRX-3). The human SPTRX-3 gene maps at 9q32, only 50 kb downstream from the TRX-1 gene from which it probably originated as genomic duplication. Therefore, human SPTRX-3 protein comprises a unique thioredoxin domain displaying high homology with the ubiquitously expressed TRX-1. Among the tissues investigated, Sptrx-3 mRNA is found exclusively in the male germ cells at pachytene spermatocyte and round spermatid stages. Light and electron microscopy show SPTRX-3 protein to be predominately located in the Golgi apparatus of pachytene spermatocytes and round and elongated spermatids, with a transient localization in the developing acrosome of round spermatids. In addition, increased levels of SPTRX-3, possibly caused by overexpression, are observed in morphologically abnormal human spermatozoa from infertile men. In addition, SPTRX-3 is identified as a novel postobstruction autoantigen. In this report, we propose that SPTRX-3 can be used as a specific marker for diverse sperm and testis pathologies. SPTRX-3 is the first thioredoxin specific to the Golgi apparatus, and its function within this organelle might be related to the post-translational modification of proteins required for germ cell-specific functions, such as acrosomal biogenesis.

Thioredoxins compose a growing family of proteins that participate in different cellular processes via redox-mediated reactions. We report here the cloning, developmental expression, and location of murid Sptrx-2. Mouse and rat SPTRX-2 proteins display a high homology to their human ortholog in the thioredoxin and NDP kinase domains, and the coding genes are located at syntenic positions. Northern blotting and in situ hybridization confirmed the testis-specific expression of murine Sptrx-2 mRNA, mostly in round spermatids. Immunohistochemical analysis of the 19 steps of rat spermiogenesis showed that SPTRX-2 expression becomes prominent in the cytoplasmic lobe of step 15-18 spermatids and diminishes in step 19 just before spermiation. However, in the spermatid tail, SPTRX-2 immunoreactivity increased from step 15 to 19 and was confined to the principal piece. By immunogold electron microscopy, SPTRX-2 was first detected scattered throughout the cytoplasm of the axoneme in step 14-15 spermatids, but began to be incorporated by step 16 into the fibrous sheath (FS). During steps 17-18, the labeling increased over the ribs and columns of the assembled FS. It peaked in step 19 and remained in the FS of epididymal spermatozoa. Immunoblots of isolated FS obtained from spermatozoa confirmed that SPTRX-2 is an integral component of the FS and a post-obstruction autoantigen in vasectomized rats. Our data indicate that SPTRX-2 incorporation into the FS lags well behind FS assembly, suggesting it is required during the final stages of sperm tail maturation in the testis and/or epididymis, where extensive disulfide bonding of FS proteins occurs.

Thioredoxins (Trx) are a class of small multifunctional redox-active proteins found in all organisms. Recently, we reported the cloning of a mitochondrial thioredoxin, Trx2, from rat heart. To investigate the biological role of Trx2 we have isolated the human homologue, hTrx2, and generated HEK-293 cells overexpressing Trx2 (HEK-Trx2). Here, we show that HEK-Trx2 cells are more resistant toward etoposide. In addition, HEK-Trx2 are more sensitive toward rotenone, an inhibitor of complex I of the respiratory chain. Finally, overexpression of Trx2 confers an increase in mitochondrial membrane potential, DeltaPsi(m). Treatment with oligomycin could both reverse the effect of rotenone and decrease the membrane potential suggesting that Trx2 interferes with the activity of ATP synthase. Taken together, these results suggest that Trx2 interacts with specific components of the mitochondrial respiratory chain and plays an important role in the regulation of the mitochondrial membrane potential.

Thioredoxins are proteins that participate in different cellular processes via redox-mediated reactions. For humans, we have recently described two novel members of this family that display a male germ cell specific expression pattern, named spermatid specific thioredoxin (Sptrx-1 and Sptrx-2 respectively). We report here the cloning and characterization of the mouse Sptrx-1 gene and protein, which are similar to those described for the human orthologue. The mouse Sptrx-1 open reading frame encodes for a protein of 462 aa composed of an N-terminal repetitive domain of a 15 residue motif followed by a C-terminal domain typical of thioredoxins. The mouse Sptrx-1 gene sequence is interrupted by only one intron of 525 bp located in the 5'-UTR, and using fluorescence in-situ hybridization we have mapped its chromosomal location to 17E1.2-1.3. Northern blot analysis identified the testis as the only tissue expressing mouse Sptrx-1 mRNA, and by in-situ hybridization we found a strong labelling in the testicular seminiferous tubules, mostly in the round spermatids. Affinity purified antibodies against human Sptrx-1 crossreacted well with the mouse protein confirming its expression in seminiferous tubules at the later stages of spermiogenesis. Recombinant mouse Sptrx-1 displayed protein disulphide reducing activity in an enzymatic assay coupled to NADPH and thioredoxin reductase. The availability of the mouse Sptrx-1 gene sequence is the first step towards the generation of knock-out mice, whose characterization will provide significant information regarding the in-vivo function of Sptrx-1 and its possible implication in several sperm anomalies.