Functional deletion of Txndc2 and Txndc3 increases the susceptibility of spermatozoa to age-related oxidative stress

Free Radical Biology and Medicine 65 (2013) 872–881

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Original Contribution

Functional deletion of Txndc2 and Txndc3 increases the susceptibility of spermatozoa to age-related oxidative stress T.B. Smith a,1, M.A. Baker a,1, H.S. Connaughton a, U. Habenicht b, R.J. Aitken a,n a Reproductive Science Group, Priority Research Centre in Reproductive Science, School of Environmental and Life Sciences, Discipline of Biological Sciences, University of Newcastle, Callaghan, NSW 2308, Australia b TRG Gynecology & Andrology and Male Health Care Research, Bayer Schering Pharma AG, Berlin, Germany

art ic l e i nf o

a b s t r a c t

Article history: Received 25 November 2012 Received in revised form 26 April 2013 Accepted 15 May 2013 Available online 23 May 2013

Oxidative stress in the male germ line is known to be a key factor in both the etiology of male infertility and the high levels of DNA damage encountered in human spermatozoa. Because the latter has been associated with a variety of adverse clinical outcomes, including miscarriage and developmental abnormalities in the offspring, the mechanisms that spermatozoa use to defend themselves against oxidative stress are of great interest. In this context, the male germ line expresses three unique forms of thioredoxin, known as thioredoxin domain-containing proteins (Txndc2, Txndc3, and Txndc8). Two of these proteins, Txndc2 and Txndc3, retain association with the spermatozoa after spermiation and potentially play an important role in regulating the redox status of the mature gamete. To address this area, we have functionally deleted the sperm-specific thioredoxins from the male germ line of mice by either exon deletion (Txndc2) or mutation of the bioactive cysteines (Txndc3). The combined inactivation of these Txndc isoforms did not have an overall impact on spermatogenesis, epididymal sperm maturation, or fertility. However, Txndc deficiency in spermatozoa did lead to age-dependent changes in these cells as reflected by accelerated motility loss, high rates of DNA damage, increases in reactive oxygen species generation, enhanced formation of lipid aldehyde–protein adducts, and impaired protamination of the sperm chromatin. These results suggest that although there is considerable redundancy in the systems employed by spermatozoa to defend themselves against oxidative stress, the sperm-specific thioredoxins, Txndc2 and Txndc3, are critically important in protecting these cells against the increases in oxidative stress associated with paternal age. & 2013 Published by Elsevier Inc.

Keywords: Spermatozoa Txndc Oxidative stress Free radicals

Oxidative stress is believed to be a key factor in the etiology of defective sperm function in approximately 40% of infertile males [1]. Such a high figure is indicative of the fact that spermatozoa are particularly susceptible to oxidative damage because they possess an abundance of substrates for free radical attack, including DNA, proteins, and a lipid profile dominated by highly unsaturated fatty acids [2]. Furthermore, spermatozoa are distinguished from somatic cells by virtue of a highly restricted cytoplasmic space in which to harbor the antioxidant enzymes that protect most cell types from oxidative stress. One of the main antioxidant defense systems known to be present in the male reproductive tract comprises members of the

Abbreviations: Txndc, thioredoxin domain-containing; ROS, reactive oxygen species; Prdx, peroxiredoxin; Gpx5, glutathione peroxidase 5; BWW, Biggers, Whitten, and Whittingham medium; PVA, polyvinyl alcohol; DHE, dihydroethidium; MSR, MitoSOX red; 4HNE, 4-hydroxynonenal n Corresponding author. Fax: +61 4921 6308. E-mail address: [email protected] (R.J. Aitken). 1 These authors contributed equally to this paper. 0891-5849/$ - see front matter & 2013 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.freeradbiomed.2013.05.021

thioredoxin–glutaredoxin family of proteins. Thus, glutathione, thioredoxin, peroxiredoxin, and glutathione peroxidase all play important roles in scavenging reactive oxygen species (ROS) and the maintenance of a balanced intracellular redox state [2,3]. To demonstrate the importance of these systems, four knockout animal models have previously been produced, targeting peroxiredoxin 4 (Prdx4), glutathione peroxidase 4 (Gpx4), glutathione peroxidase 5 (Gpx5), and a double knockout of Gpx4 and Gpx5 [4–7]. Interestingly, the Gpx5 knockout mice showed no difference in fertilization rates compared with wild-type controls; however, males over 1 year of age exhibited high levels of oxidative DNA damage in the spermatozoa, impaired nuclear DNA compaction, and an increased incidence of miscarriages and developmental defects after mating to wild-type females [4]. Deletion of Gpx4 or the double knockout of Gpx4 and Gpx5 was also found to result in spermatozoa exhibiting impaired chromatin compaction and high levels of nuclear DNA damage [6,7]. Furthermore, in the double knockout the epididymis was shown to mount a compensatory antioxidant response featuring a dramatic upregulation of key protective enzymes including protein disulfide isomerases, glutathione S-transferases, and

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members of the thioredoxin/peroxiredoxin system [7]. Further evidence suggesting that the thioredoxin/peroxiredoxin system plays a critical role in preserving the integrity of the male germ line was provided by Prdx4 knockout mice [5], which demonstrated oligozoospermia associated with higher levels of oxidative damage in the male germ line compared with their wild-type counterparts. In addition to the somatic expression of thioredoxins [8], spermatozoa have their own unique versions of these redox proteins, in keeping with their particular need for efficient protection against oxidative stress. These germ-line thioredoxins include sperm-specific thioredoxin-1, Sptrx-1 [9,10], Sptrx-2 [11,12], and Sptrx-3 [13]. These proteins are also known as thioredoxin domain-containing 2 (Txndc2), thioredoxin domaincontaining 3 (Txndc3), and thioredoxin domain-containing 8 (Txndc8), respectively, and, because this is the preferred nomenclature, it is used throughout this article. Txndc2 comprises two distinct regions including an N-terminal domain with 23 highly conserved repetitions of 15 amino acids and a C-terminal thioredoxin domain [9,14]. Northern blot and in situ analyses both demonstrate that Txndc2 is expressed only in the testis, being found at both the round and the elongating spermatid stage of spermatogenesis. Immunogold labeling demonstrated that Txndc2 also localizes to spermatozoa, specifically being present in the postacrosomal region and along the neck and principal piece of human spermatozoa. This pattern of localization prompted the authors to suggest that Txndc2 may be involved in the stabilization of flagellar cysteines during sperm maturation [9,14]. Txndc3 is composed of an N-terminal thioredoxin domain and three C-terminal nucleoside diphosphate (NDP) kinase domains and is incorporated into the ribs and longitudinal columns of the fibrous sheath in the final stages of spermatid development. Txndc3 remains as an integral component of the fibrous sheath in mature spermatozoa and also represents a postobstruction autoantigen in vasectomized rats [12]. In situ analysis of human testis has also demonstrated that a third representative of the sperm thioredoxin family (Txndc8) is present in the male germ line. Immunohistochemical analyses demonstrated that this protein was present in the Golgi apparatus of pachytene spermatocytes and spermatids and exhibited a transient localization in the developing acrosome of round spermatids [13]. Once the sperm acrosome is formed, Txndc8 accumulates in the cytoplasm of elongating spermatids, before being discharged into the residual bodies and phagocytosed by Sertoli cells. In defective human spermatozoa retaining excess residual cytoplasm this particular sperm-specific thioredoxin is retained and therefore has some diagnostic value as a marker for incomplete cytoplasmic extrusion [13]. However, because Txndc8 is not retained by normal spermatozoa after spermiation, it cannot be significantly involved in the physiological regulation of sperm function. To gain more insights into the role of the sperm-specific thioredoxin system in the control of sperm function, transgenic mice have been generated in which the two Txndc proteins that retain association with the differentiated gamete (Txndc2 and Txndc3) have been functionally deleted. In the case of Txndc2, this was achieved by generating a knockout mouse through the deletion of an entire exon, whereas for Txndc3, the active thioredoxin domain of this molecule was mutated such that the amino acid residues WCGPC found on exon 4 were mutated to produce WSGPS (two cysteine-to-serine mutations). In this manner, the three NDP kinase domains of Txndc3 remained functional, allowing us to study the function of the Txndc3 thioredoxin domain in isolation. These mice were then crossed to generate animals in which these sperm-specific thioredoxins were functionally deleted. The phenotypic analysis of these animals provides

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definitive proof that these highly specialized enzymes play an important role in protecting the male germ line against oxidative stress.

Materials and methods Reagents All reagents were purchased from Sigma–Aldrich unless otherwise stated. Warmed, fresh Biggers, Whitten, and Whittingham (BWW) medium was used for all experiments and is composed of 91.5 mM NaCl, 4.6 mM KCl, 1.7 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 25 mM NaHCO3, 5.6 mM D-glucose, 0.27 mM sodium pyruvate, 44 mM sodium lactate, 20 mM Hepes buffer supplemented with 1 mg/ml polyvinyl alcohol (PVA), 5 U/ml penicillin, and 5 mg/ml streptomycin, and the osmolarity was kept between 290 and 310 mOsm/kg [15]. All fluorescent probes were purchased from Molecular Probes, Inc. (Eugene, OR, USA). Creation of the Sptrx knockout lines Transgenic mice were produced in the animal facility at TaconicArtemis GmbH (Cologne, Germany) in microisolator cages (Tecniplast Sealsafe, Buguggiate, Italy). The mouse Txndc2 gene was subcloned from a genomic C57BL/6 RP23 BAC library and recloned into the basic targeting vector harboring loxP sites flanking exon 3 of Txndc2, an FRT flanked Neo cassette for positive selection, and a ZsGreen cassette for counterselection (Fig. 1A). The mouse Txndc3 gene was subcloned with the conserved WCGPC site mutated to WSGPS and recloned into the basic targeting vector (Fig. 1D). After administration of hormones, superovulated Balb/c Ola/Hsd females were mated with Balb/c Ola/Hsd males. Blastocysts were isolated from the uterus at 3.5 days postcoitus (dpc). For microinjection, blastocysts were placed in a drop of Dulbecco's modified Eagle's medium with 15% fetal calf serum under mineral oil. A flat-tip, piezo-actuated microinjection pipette with an internal diameter of 12–15 μm was used to inject 10–15 targeted C57BL/6 N.tac embryonic stem (ES) cells into each blastocyst. After recovery, eight injected blastocysts were transferred to each uterine horn of 2.5-dpc pseudopregnant NMRI females. Chimerism was determined according to coat color (black/white, indicating the contribution of ES cells to the Balb/c host). One homologously recombined clone harboring the targeted allele was used for the generation of chimeric mice by blastocyst injection. Highly chimeric mice were bred to C57BL/6 females, and offspring heterozygous for the targeted allele were identified by Southern blot. To eliminate the selection marker and, in the case of Txndc2, exon 3, mice heterozygous for the targeted allele were bred with mice carrying one copy of the Cre recombinase transgene under the control of the ROSA26 locus (C57BL/6-Gt[ROSA]26Sortm16[Cre] Arte). The resulting offspring, heterozygous for the null allele (C57BL/6-Gpx5tm1115_.2 Arte), were backcrossed with C57BL/6 mice to eliminate the Cre recombinase transgene. Wild-type and mutant animals were derived from heterozygous intercrosses and were devoid of the Cre recombinase transgene (a potential source of interference with the mouse genome). Mice were maintained on a 14-h light/10-h dark cycle and provided with food and water ad libitum. All animal procedures were run according to German animal welfare laws and with the permission of the district government of Berlin. Once housed in the University of Newcastle housing facility, mice were maintained under conditions of controlled temperature (22 1C) and a 12-h light/12-h dark cycle with food and water ad libitum. From this point on, all animal care and procedures were approved according to the University of Newcastle animal ethics guidelines.

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Fig. 1. Generation of Txndc2- and Txndc3-deficient mice. (A) Schematic representations of the mouse wild-type Txndc2 allele, the targeting vector plasmid, the Txndc2 allele after homologous recombination with the targeting vector, and the conventional allele after Cre-mediated deletion of exon 3. (B) Agarose gel analysis of PCR products amplified from genomic DNA extracted from ear punches of Txndc2+/+, Txndc2+/−, and Txndc2−/− mice using multiple primers to amplify both wild-type and knockout alleles. Internal control primers were also included to amplify a product of 585 bp. (C) Schematic representations of the mouse wild-type Txndc3 allele, the targeting vector plasmid, the Txndc3 allele after homologous recombination with the targeting vector, and the conventional allele after Flp-mediated point mutation. (D) Agarose gel analysis of PCR products amplified from genomic DNA extracted from ear punches of Txndc3+/+, Txndc3+/−, and Txndc3−/− mice using multiple primers to amplify both wild-type and knockin alleles. Internal control primers were also included to amplify a product of 585 bp.

Genotyping of mice by PCR

Cauda sperm extraction by perfusion

Genomic DNA was extracted from mouse ear punches into 100 ml of extraction buffer containing 50 mM Tris–HCl, 2% SDS, 10 mM EDTA, 0.75 M NaCl, and 100 mg/ml proteinase K overnight at 56 1C. Supernatant was precipitated in 100% v/v isopropanol, the pellet washed in 70% v/v ethanol, and genomic DNA resuspended in 50 ml of TE buffer by heating at 65 1C. Genotyping of Txndc2 was performed by PCR in a single reaction using the following primers: wild-type band 1, Fw 5′-CATTCATCAGATGCCTTCATGG-3′, Rev 5′-GAAGAGTCTGAGTTCCCGCAG-3′ (a 457-bp product); wild-type band 2, Fw 5′-CTTGGAGGTGGACACTGAGGA-3′, Rev 5′-CCATGATGAAATCCATGCCT-3′ (a 703-bp product); conventional KO band Fw 5′-CATTCATCAGATGCCTTCATGG-3′, Rev 5′-CCATGATGAAATCCATGCCT-3′ (a 393-bp product). Genotyping of Txndc3 was performed by PCR with the following primers: Fw 5′-GGGATTAAATCTCACCGTCAAATGC-3′, Rev 5′-GCACTTGCTGGCAGGCATGAT-3′ (KI band at 346 bp and a wild-type band at 267 bp). An internal control consisting of the Fw 5′-GAGACTCTGGCTACTCATCC-3′ and Rev 5′-GTCCCCAGCTCTTGCTGAAGG-3′ primers (a 585-bp product) was included in both Txndc2 and Txndc3 genotyping PCRs. In all cases, PCR amplification was performed for 35 cycles (5 min at 95 1C, 30 s at 95 1C, 30 s at 61.4 1C, 1 min at 72 1C, 10 min at 72 1C) using Taq DNA polymerase (Invitrogen, Carlsbad, CA, USA). PCR products were analyzed on 1% agarose gels, using UV light after ethidium bromide staining.

Adult male mice were asphyxiated via CO2 inhalation and the epididymides and testes removed immediately, blotted free of blood, and placed in prewarmed water-saturated mineral oil. Pure suspensions of spermatozoa were obtained from the caudal region of the epididymis by backflushing and the perfusate was allowed to disperse for 10 min at 37 1C in 1 ml of BWW containing 1 mg/ml PVA, followed by light centrifugation at 400 g for 3 min to remove epididymal fluids, and the pellet was resuspended in 1 ml BWW/ PVA. All experiments were performed promptly, while keeping the spermatozoa at 37 1C.

Computer-aided sperm assessment (CASA) The movement characteristics of the spermatozoa were examined with a Hamilton Thorne IVOS motility analyzer, version 10.5 K, at an incubation temperature of 37 1C. Each sample was loaded into 20-mm-deep MicroCell slides (Microm, Thame, UK) and the analysis was performed on five random fields from each sample. At least 200 cells were analyzed. The settings for mouse spermatozoa were negative phase-contrast optics, recording rate 60 frames/s, minimum contrast 50, minimum cell size 4 pixels; threshold values for progressively motile spermatozoa were VAP (average path velocity) 450 mm/s and straightness 4 80%; slow

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cell VAP cutoff, 7.4 mm/s, slow cell straight-line velocity cutoff, 6.6 mm/s. Diff-Quick morphological staining An aliquot of fresh caudal spermatozoa was smeared on Polysine slides (Menzel–Glaser, Braunschweig, Germany) and stained with Diff-Quick (Lab Aids, Narrabeen, Australia), following the manufacturer's instructions. Slides were mounted in DPX and viewed with an Axio Imager A1 fluorescence microscope (Carl Zeiss Microimaging, Thornwood, NY, USA) using phase-contrast optics, and images were taken on an Olympus DP70 camera (Olympus America, Centre Valley, PA, USA). Dihydroethidium staining Dihydroethidium (DHE) and the vitality stain Sytox green (SyG) were diluted in BWW/PVA and added to 5  105 spermatozoa in a final volume of 50 μl, comprising 45 μl of purified cauda sperm suspension and 5 μl DHE/SyG mixture, equating to final concentrations of 2 and 0.05 μM, respectively. The cells were then incubated in the dark at 37 1C for 15 min and dispensed onto a warmed microscope slide and the resulting red and green fluorescence was analyzed immediately using an Axio Imager A1 fluorescence microscope (Carl Zeiss) under fluorescence optics (470 nm excitation). At least 200 cells were counted and scored as live negative, live positive, or dead. MitoSOX red Approximately 5  105 cells were stained with MitoSOX red (MSR) at a final concentration of 2 mM together with the vitality stain SyG (0.05 mM) for 15 min at 37 1C, under conditions where they were shielded from light. After incubation, 5 ml of cells were placed on a warmed microscope slide and the resulting red and green fluorescence was analyzed immediately using an Axio Imager A1 fluorescence microscope (Carl Zeiss) under fluorescence optics (470 nm excitation). At least 200 cells were counted and scored as live negative, live positive, or dead. JC-1 To assess mitochondrial function, 45 ml of sperm suspension (containing 5  105 cells) was co-incubated with 5 ml of JC-1 to give a final concentration of 20 mM. After incubation with JC-1 for 15 min at 37 1C spermatozoa were placed on a prewarmed microscope slide and immediately scored as having high mitochondrial membrane potential (orange/yellow) or low mitochondrial membrane potential (green) using an Axioplan 2 microscope (Carl Zeiss). Alkaline comet assay Slides were coated with agarose containing spermatozoa and then were immersed in a lysing solution of 2.5 M NaCl, 100 mM EDTA, 10 mM Trizma, pH 10, supplemented with 10 mM dithiothreitol and 1% Triton X-100 for 1 h at 37 1C. Slides were placed in a horizontal gel electrophoresis tank (Millipore, Billerica, MA, USA) with electrophoresis buffer (1 mM EDTA, 300 mM NaOH, pH 13) and cooled to 4 1C for 20 min to allow the DNA to unravel. Electrophoresis was conducted at 25 V for 5 min at 350–400 mA. After electrophoresis, the slides were neutralized in a buffer containing 0.4 M Trizma in ddH2O (pH 7.5) for 5 min, stained with 20 mg/ml ethidium bromide, and viewed immediately under fluorescence, and images were captured using an Olympus DP70 camera (Olympus America). At least 100 cells were analyzed per

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slide using Comet Assay IV version 4.2 (Perceptive Instruments, Haverhill, Suffolk, UK), recording tail intensity and tail moment, but excluding cells that did not lyse completely. All samples were run in duplicate. CMA3 Approximately 5  105 caudal spermatozoa were fixed with 2% paraformaldehyde for 15 min at 4 1C, washed for 5 min, and stored at 4 1C in 0.1 M glycine for a maximum of 1 week. An aliquot of the sperm suspension was settled onto a poly-L-lysine coverslip in a humidified chamber for at least 2 h at 4 1C. Sperm were permeabilized in 0.2% Triton X-100 in phosphate-buffered saline (PBS) for 15 min at 37 1C and then washed once in McIlvaines buffer. Coverslips were then stained with 25 ml of CMA3 solution (0.25 mg/ml in McIlvaines buffer) at room temperature for 20 min protected from light, washed twice, mounted with Mowiol, and sealed. At least 100 cells were scored under fluorescence and scored as positive or negative. Protein extraction and quantification For one-dimensional sodium dodecyl sulfate (SDS)–PAGE and Western blot analysis, spermatozoa were washed with PBS and the cell pellet was extracted with SDS extraction buffer (0.5% SDS, 10% sucrose in 0.1875 M Tris, pH 6.8) containing a protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany) at 100 1C for 5 min and vortexed vigorously, and the insoluble cellular debris was removed by centrifugation at 10,000 g for 15 min at 4 1C. Quantification of the isolated protein supernatant was achieved using a BCA (bicinchoninic acid) protein assay kit (Pierce, Rockford, IL, USA) according to the manufacturer's instructions. SDS–PAGE/Western blot Approximately 10 mg of protein from spermatozoa was boiled in SDS–PAGE sample buffer (SDS extraction buffer as described above supplemented with 2% 2-mercaptoethanol and bromophenol blue) for 5 min and resolved on Tris–glycine 4–20% polyacrylamide gels (NuSep, Sydney, Australia). The resolved proteins were then transferred to nitrocellulose membranes under a constant current of 300 mA for 1 h. Nitrocellulose membranes were blocked overnight in 5% skim milk powder in TBS (Tris-buffered saline; 100 mM Tris–HCl, pH 7.6, and 150 mM NaCl) (pH 7.4) supplemented with 0.1% Tween 20 (TBST). Membranes were rinsed in TBS and probed overnight with anti-4HNE (Jomar Diagnostics, Adelaide, Australia) at a concentration of 1:500, washed, and then probed for 1 h with a 1:1000 dilution of horseradish peroxidase-conjugated secondary antibody at room temperature. After a further three washes in TBST, cross-reactive proteins were visualized using an enhanced chemiluminescence kit (GE Healthcare, Piscataway, NJ, USA) according to the manufacturer's instructions. Thiol expression To assess the levels of free thiol expression in the spermatozoa these cells were lysed in SDS extraction buffer containing 2% SDS, 0.1875 M Tris, pH 6.7, 10% sucrose, and a protease inhibitor cocktail mix tablet (Roche Diagnostics). Protein concentration was subsequently determined using the Pierce BCA protein assay kit. Ten micrograms of protein in 50 ml of SDS extraction buffer was incubated with BODIPY-NEM at a final concentration of 50 mM for 30 min at room temperature in the dark; 2 mg of protein was then added 1:1 with loading buffer containing β-mercaptoethanol at a final concentration 0.2 M to reduce all nonalkylated cysteine residues. The samples were then loaded into the wells of a 4–20%

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nUView NuSep precast gel (NuSep) incorporating a proprietary fluorescent protein marker. The gels were run at 120 (stacking) and 180 V (resolving) for approximately 1.5 h in the dark before being imaged on a Typhoon Trio imager (GE Healthcare). For this purpose, the precast gel was removed from the glass plates and placed directly onto the surface of the Typhoon Trio imager. The focal plane was set at “platen” and the photomultiplier was set between 400 and 550 V with 100–200 mm resolution. A fluorescence image was acquired by using the green laser (λ 532 nm) in combination with the 526SP filter. The acquired image was then sent to Imagequest (GE Healthcare) for analysis.

Statistics All experiments were replicated at least three times on independent samples and the results analyzed by one- and two-way ANOVA using the SuperANOVA program (Abacus Concepts, Piscataway, NJ, USA) on a MacIntosh G4 Powerbook computer; post hoc comparison of group means was by Fisher's protected least significant difference. Paired comparisons were conducted using

a paired t test using the Statview program (Abacus Concepts). Differences with a P value of o0.05% were regarded as significant.

Results Txndc2 knockout strategy and verification As shown in Fig. 1A, the Txndc2 gene is composed of three exons spanning 4.7 kb on mouse chromosome 17. A targeting vector containing the Neo cassette and exon 3 sequence flanked by loxP sites was generated (Fig. 1A). Embryonic stem cells from C57BL/6 mice, transformed by the targeting vector and recombination, were screened by Southern blot (data not shown). After injection of positive recombinant ES cells into blastocysts and reintroduction of the blastocysts into female mice prepared for gestation, chimeric mice were created. First, matings between heterozygous Txndc2+/− mice and Cre+/+ mice were performed to generate mice free of the Neo cassette and exon 3. Intercrosses between Txndc2+/−[Cre] mice were then carried out to eliminate the Cre allele. When PCR analysis was performed on transgenic

Fig. 2. Impact of Txndc2 and Txndc3 deficiency on fertility. (A) The numbers of pups/litter were comparable between wild-type, Txndc2+/−, Txndc2−/−, Txndc3+/−, Txndc3−/−, and double-transgenic males ages 3–9 months. Hematoxylin and eosin staining of (B) testes and (C) caudal epididymal tissue sections showed no obvious aberrations in the reproductive organs of wild-type, Txndc2−/−, Txndc3−/−, and double-transgenic males. (D) Mature spermatozoa obtained from the cauda epididymides and stained with DiffQuick failed to reveal the presence of noticeable morphological irregularities in the terminally differentiated gametes.

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animals, we detected the presence of both wild-type and deleted alleles in heterozygous animals and no wild-type allele in homozygous Txndc2−/− animals, indicating that exon 3 had been successfully deleted (Fig. 1B). Txndc3 mutation strategy and verification As shown in Fig. 1C, the Txndc3 gene is composed of 17 exons spanning 65 kb on mouse chromosome 17. A targeting vector containing the Neo cassette flanked with a Flp-recombinase site and exon 4 with the mutated sequence (C39S and C42S) was constructed. Embryonic stem cells from C57BL/6 mice were transformed by the targeting vector and screened for recombination by Southern blot (data not shown). Positive clones were injected into blastocysts and reintroduced back into female mice that were prepared for gestation. First, matings between heterozygous Txndc3+/− C39S C42S mutant mice and Flp+/+ mice were performed to generate mice free of the Neo cassette. The mice were then bred as for Txndc2. When PCR analysis was performed on transgenic animals, we detected the presence of both wild-type and mutated alleles in the Txndc3+/− heterozygotes but only the mutated allele in the Txndc3−/− homozygotes, as shown in Fig. 1D. Evaluation of fertility and reproductive histology The aim of this project was to understand the function of thioredoxins in terms of sperm physiology. Given that Txndc2 and Txndc3 are both testis-specific transcripts, we began our investigation by looking at the reproductive consequences of either knocking out Txndc2 or mutating Txndc3 in isolation and then examined the double-transgenic cross; all of the mice used in these mating trials were 3–9 months of age. We found that neither the knockout of Txndc2 nor the functional disruption of Txndc3 had any appreciable effect on either pregnancy rates or the average number of pups born, suggesting that the thioredoxin system in the male reproductive tract may exhibit significant functional redundancy (Fig. 2A). To interrogate this possibility more thoroughly, we next created a double-transgenic mouse line, by crossing the Txndc2−/− mice with the Txndc3 homozygous mutants. As shown (Fig. 2A), no observable differences in litter size were observed

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when comparing the double-transgenic males with wild-type mice. Furthermore, the ratio of male to female pups was unaffected by the combined disruption of Txndc2 and Txndc3 (data not shown) and the preweaning mortality rate was comparable between the wild-type litters (2.8%), Txndc2−/− litters (2.9%), and Txndc3−/− mutant litters (2.7%) and the double-transgenic mice (3.0%). Histological analysis of wild-type, Txndc2−/− and Txndc3−/− mutant, and double-transgenic testes differentially stained with hematoxylin and eosin revealed no obvious aberrations in the spermatogenic process (Fig. 2B). The cauda epididymis of each genotype also appeared normal, with no clear distinction in sperm concentration or histology (Fig. 2C). Spermatozoa obtained from the cauda epididymis were stained with Diff-Quick to assess their morphology. Again no apparent differences were observed between the genotypes (Fig. 2D). Functional disruption of Txndc leads to an age-dependent loss of sperm motility Because oxidative stress in the male reproductive tract increases with age [16] we determined whether the anticipated decrease in antioxidant protection precipitated by the functional deletion of Txndc2 and Txndc3 might be associated with a reproductive phenotype in aging males. We began by using CASA technology to examine the movement characteristics of spermatozoa in relation to age, because sperm motility is known to be particularly sensitive to oxidative stress [17]. At 6 months of age, total motility and progressive motility in the double-transgenic mice were comparable to those of the wild type (Fig. 3A). However, by 12 months of age, total motility of spermatozoa from the double-transgenic animals had decreased by around 15% compared to wild type (P o 0.05; Fig. 3A); at 18 months of age, the decline in progressive motility had doubled to 30% and by 24 months of age total motility and progressive motility had declined to approximately half that of the wild type, such that only 10% of double-knockout spermatozoa displayed forward, progressive movement (P o 0.001; Fig. 3B). These changes in sperm motility were not accompanied by any overt changes in sperm number or morphology.

Fig. 3. Motility declines with increasing age in Txndc-deficient spermatozoa. (A) CASA analysis revealed a significant decline in total motility from 12 months of age in double-transgenic (DT) spermatozoa, becoming progressively more pronounced at 18 and 24 months of age. (B) Progressive motility also declined with age in DT spermatozoa, being statistically significant at 18 and 24 months of age. Motility analysis was performed on six animals. WT, wild type. Bars represent 7 SEM. nP o 0.05; nn P o 0.01; nnnP o 0.001.

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Functional disruption of Txndc leads to an age-dependent increase in ROS generation Analysis of spermatozoa from the 12- and 18-month-old double-transgenic mice stained with a viability stain, Sytox green, confirmed that the motility decline with advancing age was not associated with a loss of cell viability (Fig. 4A). Although ATP is predominantly produced via glycolysis in murine spermatozoa and not oxidative phosphorylation, a loss of mitochondrial membrane potential is an early marker of the apoptotic pathway that leads to rapid motility loss in human spermatozoa [18]; however, no loss of mitochondrial membrane potential was observed in Txndc doubletransgenic mice (Fig. 4B). Despite the normality of the mitochondrial membrane potential, a significant percentage of spermatozoa at 18 months of age were found to exhibit high levels of ROS, as measured by DHE (Fig. 4C; P o 0.01) and MSR (Fig. 4D; P o 0.05), compared to the wild-type controls. Thus, these results indicated that spermatozoa from aging mice lacking functional Txndc2 and Txnd3 were producing more superoxide anion (O2d−) from intracellular sites, including their mitochondria. 4HNE adducts are increased in Txndc−/− mice but thiol expression is unchanged If the spermatozoa of aging Txndc−/− mice were producing more O2d− as suggested by the increased DHE and MSR signals, then it would be predicted that these same cells would also be experiencing an increased level of lipid peroxidation. A recent study [19] has revealed that the electrophilic by-products of lipid peroxidation, such as 4-hydroxynonenal, become adducted to enzymes in the sperm mitochondria, initiating a self-perpetuating cycle of O2d− generation from these organelles, thereby initiating an apoptotic cascade that invariably leads to motility loss. To assess the presence of 4HNEadducted proteins in Txndc-deficient mice, SDS-extracted protein lysates from 2-year-old wild-type and Txndc2 and Txndc3 doubletransgenic spermatozoa were subjected to SDS–PAGE, blotted, and probed with an antibody against 4HNE. Although a number of bands corresponding to 4HNE-adducted proteins were identified in the wildtype and double-transgenic lysates (Fig. 4E), the level of adduction appeared to be higher in the latter. To confirm this point, we used densitometry to compare the intensity of the 4HNE staining with the α-tubulin loading controls (Fig. 4F). This analysis corroborated the visual interpretation of this blot in revealing that the 4HNE signal was twice as intense in the Txndc-deficient mice as in the wild type. An analysis of free thiol expression in 2-year-old control and Txndc−/− mice did not reveal any major changes in thiol expression under these conditions (Fig. 4G). Txndc deficiency affects sperm DNA integrity and chromatin protamination Given the increased levels of ROS generation and lipid peroxidation observed in the spermatozoa of double-transgenic mice, we next examined the status of sperm chromatin in these animals and age-matched controls. For this purpose we employed an alkaline comet assay (Fig. 5A) to measure the integrity of sperm nuclear DNA and the CMA3 assay to determine the level of sperm chromatin protamination. This analysis detected a small but highly significant age-related decrease in the levels of chromatin protamination in the Txndc-deficient mice at 18 months of age (P o 0.001) but not 6 months earlier (Fig. 5B), in keeping with the late onset of ROS generation and high levels of lipid peroxidation (Fig. 4C–F). In contrast, the comet assay revealed an increase in DNA damage in the spermatozoa of Txndc-deficient mice that was statistically significant (P o 0.001) at both 12 and 18 months of age.

Fig. 4. Impact of Txndc deficiency on sperm quality. (A) Sytox green revealed no loss of viability in double transgenic (DT) spermatozoa at 12 and 18 months of age. (B) Similarly, mitochondrial membrane potential was comparable between wildtype (WT) and DT spermatozoa at 12 and 18 months, measured by JC-1. (C) The percentage of live spermatozoa generating O2d− from the cytosol was increased in DT spermatozoa at 18 months of age, as detected by DHE. (D) The percentage of spermatozoa generating O2d− from their mitochondria was also significantly elevated in DT males at 18 months of age. (E) Western blot analysis revealed bands corresponding to 4HNE-adducted proteins in both wild-type and DT spermatozoa at 24 months of age. (F) Densitometry analysis as a percentage of the tubulin loading control revealed a significant increase in the density of 4HNE-adducted proteins in DT spermatozoa. (G) Similar patterns of thiol expression in wild-type and double-transgenic animals. Experiments for (A)–(D) were performed on six animals and for (E), (F), and (G) on three animals. Bars represent 7 SEM. nP o 0.05; nn P o 0.01.

Discussion ROS represent a two-edged sword as far as spermatozoa are concerned. On one hand a low level of ROS is required to drive the tyrosine phosphorylation events associated with sperm capacitation [20,21]. On the other, spermatozoa are very vulnerable to

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Fig. 5. Impact of Txndc deficiency on DNA integrity in caudal epididymal spermatozoa. (A) A representative image of the alkaline comet assay optimized for caudal mouse spermatozoa. (B) CMA3 staining revealed aberrations in the efficiency of protamination at 12 and 18 months of age in DT spermatozoa, which were statistically significant at 18 months. (C) Comet analysis revealed significant increases in DNA damage in the double-transgenic animals at both 21 and 24 months compared with controls. WT, wild type; DT, double transgenic. Experiments were performed on six animals. Bars represent 7 SEM. nnnP o 0.001.

oxidative stress to the point that oxidative damage seems to be a major causative factor in the etiology of male infertility [22]. It has recently been suggested that these two situations may represent a continuum in the life history of the spermatozoon [23]. The generation of ROS drives spermatozoa down a pathway of capacitation until a point is reached at which the limited antioxidant defenses offered by these cells are finally overwhelmed and the spermatozoa enter of state of oxidative stress, culminating in activation of the intrinsic apoptotic cascade and cell death. Given this propensity for ROS generation it is not surprising that spermatozoa have invested in multiple, enzymatic antioxidant defense mechanisms including the superoxide dismutase, glutathione/glutathione peroxidase, and thioredoxin/peroxiredoxin systems [24–26]. Gpx and Prdx are thought to be the main mechanisms for mediating fine adjustments to H2O2 exposure during epididymal maturation, as revealed by the oxidative stress phenotypes reported in the spermatozoa of Prdx4, Gpx5, and

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Gpx4/Gpx5 double-knockout animals [4–7]. When H2O2 is reduced by sperm Prdx, using reducing equivalents provided by thioredoxin, the thiols at the active site are deactivated in a dosedependent manner [27]. Only then can they be reactivated by the thioredoxin/thioredoxin reductase system [28]. The exception is peroxiredoxin 6, which can be regenerated by glutathione [29]. A comparison of the levels of oxidized Prdx1 in the spermatozoa of infertile patients and healthy controls revealed an increase in the levels of oxidized enzyme in the former, thereby depriving these cells of antioxidant protection [30]. Two possible explanations for the persistent oxidized state of Prdx1 in infertile patients are not mutually exclusive. First, H2O2 is being consistently generated in higher amounts by the infertile subjects’ spermatozoa, in keeping with numerous previous studies reporting increased ROS generation by defective sperm [3,22]. Second, defects in the thioredoxin/ thioredoxin reductase system in the infertile samples may be affecting the rate of regeneration of Prdx1. The human thioredoxin/thioredoxin reductase system has previously been demonstrated to be sensitive to alkylating agents, including dinitrohalobenzenes [31], acrylamide [32], N-ethylmaleimide [33], and aryl chloroethylureas [34]. Interestingly, it has also been shown that 4HNE, a natural by-product of lipid peroxidation found in high concentrations in defective spermatozoa [19], readily alkylates thioredoxin and thioredoxin reductase [35]. Deactivation of the thioredoxin system by such means would be anticipated to upset the redox balance in spermatozoa, inducing a state of oxidative stress and consequently a loss of sperm function, via the self-perpetuating cycle of lipid aldehyde generation and mitochondrial ROS generation recently reported in human spermatozoa [19]. According to this model spermatozoa would be expected to respond to thioredoxin depletion by increasing mitochondrial ROS production because electrophilic lipid aldehydes generated as a consequence of lipid peroxidation have been shown to bind to mitochondrial proteins involved in electron transport, including succinic acid dehydrogenase [19]. Adduction of these proteins perturbs the regulated flux of electrons through the mitochondrial electron transport chain leading to electron leakage and the adventitious formation of superoxide anion (Fig. 4C and D). Conversely, oxidative stress is not a known trigger for NADPH oxidase activation in spermatozoa. As a counterbalance to ROS-mediated attack, the male germ line features a number of antioxidant enzymes designed to protect these cells from oxidative stress. The high level of redundancy inherent in this defense system may explain why there was no immediate consequence to the functional deletion of spermspecific thioredoxins (Txndc2 and Txndc3) from the male germ line. Although a male reproductive phenotype did eventually emerge in the double-transgenic animals, it took time. In the first few months of life, the spermatozoa in the double transgenic mice were able to compensate for any oxidative stress arising from their lack of Txndc. However, the increased oxidative stress associated with age [16] resulted in the ultimate expression of an oxidative stress phenotype in the spermatozoa. Thus after 12 months of age the Txndc-deficient mice exhibited a significant loss of motility (Fig. 3A) and a significant increase in DNA damage (Fig. 5C), both of which are known to result from oxidative stress [3]. By 18 months, even more signs of oxidative stress had begun to surface, including significant increases in ROS generation, 4HNE adduction, and a decrease in sperm chromatin protamination. As indicated above, the increase in lipid aldehyde (4HNE) formation might have been responsible for increasing the level of oxidative stress in the spermatozoa by forming adducts with enzymes in the mitochondrial electron transport chain and triggering an increase in mitochondrial ROS [19]. The significant decrease in DNA protamination reflects the phenotype of the Gpx5-knockout mouse, which also exhibited an identical age-

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dependent change in CMA3 fluorescence [5]. This finding also echoes the human situation in which high levels of oxidative DNA damage in spermatozoa are associated with a reduction in sperm protamination as revealed by the CMA3 assay [36]. This association may mean that in all these model systems (Gpx5 knockout, Txndc double-transgenic, and male infertility) oxidative stress is experienced by spermatozoa during spermiogenesis when the chromatin is being remodeled and nuclear histones are being replaced by protamines. The DNA damage that accompanies the loss of protamination may reflect parallel nonsequential changes in spermatozoa, oxidative stress during spermiogenesis impairing chromatin protamination and simultaneously inducing oxidative damage in the DNA. Alternatively some other factor may impair chromatin protamination and render the sperm DNA vulnerable to oxidative attack at some later stage of development [36,37]. We have used BODIPY-NEM as a probe to examine the thiol status of wild-type and Txndc double-transgenic spermatozoa. By such means we hoped to determine whether the disruption of DNA compaction, and hence the increase in CMA3 staining, could reflect the 4HNE alkylation of protamines that would otherwise have been involved in creating the disulfide bridges that stabilize sperm chromatin. However, no major differences in the profile of thiols available for alkylation were evident in this analysis (Fig. 4G). Possibly a more detailed analysis of thiol status, focusing exclusively on the status of sperm chromatin, may be required to reveal changes due to Txndc depletion. Whatever mechanisms are involved, these results clearly emphasize that spermatozoa are vulnerable to oxidative attack and that the sperm-associated electron donors, Txndc2 and Txndc3, play key roles in protecting the integrity of both sperm chromatin and the cellular machinery responsible for regulating sperm motility. In summary, the production of double-transgenic mice, lacking both Txndc2 and Txndc3, produces male mice that, although fertile, display an age-dependent deterioration in sperm quality. Thus, although significant redundancy does exist in the antioxidant strategies adopted by spermatozoa, the lack of a specialized thioredoxin defense system in the mature gamete clearly compromises the ability of these cells to accommodate the increased oxidative stress associated with age.

Acknowledgments We thank Kristy Taubman for establishing the transgenic lines, Louise Hetherington for her assistance throughout this study, and Dr. Cristian O'Flaherty, McGill University, for his helpful comments on the manuscript. We also thank Schering AG for funding the initial stages of the project including the generation of the genetically modified mice. The project was completed with funding from NHMRC Program Grant 494802 and ARC Discovery Grant 110103951, for which we are also very grateful.

References [1] Tremellen, K. Oxidative stress and male infertility—a clinical perspective. Hum. Reprod. Update 14:243–258; 2008. [2] Aitken, R. J.; Curry, B. J. Redox regulation of human sperm function: from the physiological control of sperm capacitation to the etiology of infertility and DNA damage in the germ line. Antioxid. Redox Signaling 14:367–381; 2011. [3] Vernet, P.; Aitken, R. J.; Drevet, J. R. Antioxidant strategies in the epididymis. Mol. Cell. Endocrinol. 216:31–39; 2004. [4] Chabory, E.; Damon, C.; Lenoir, A.; Kauselmann, G.; Kern, H.; Zevnik, B.; Garrel, C.; Saez, F.; Cadet, R.; Henry-Berger, J.; Schoor, M.; Gottwald, U.; Habenicht, U.; Drevet, J. R.; Vernet, P. Epididymis seleno-independent glutathione peroxidase 5 maintains sperm DNA integrity in mice. J. Clin. Invest. 119:2074–2085; 2009. [5] Iuchi, Y.; Okada, F.; Tsunoda, S.; Kibe, N.; Shirasawa, N.; Ikawa, M.; Okabe, M.; Ikeda, Y.; Fujii, J. Peroxiredoxin 4 knockout results in elevated spermatogenic cell death via oxidative stress. Biochem. J. 419:149–158; 2009.

[6] Conrad, M.; Moreno, S. G.; Sinowatz, F.; Ursini, F.; Kölle, S.; Roveri, A.; Brielmeier, M.; Wurst, W.; Maiorino, M.; Bornkamm, G. W. The nuclear form of phospholipid hydroperoxide glutathione peroxidase is a protein thiol peroxidase contributing to sperm chromatin stability. Mol. Cell. Biol. 25:7637–7644; 2005. [7] Noblanc, A.; Peltier, M.; Damon-Soubeyrand, C.; Kerchkove, N.; Chabory, E.; Vernet, P.; Saez, F.; Cadet, R.; Janny, L.; Pons-Rejraji, H.; Conrad, M.; Drevet, J. R.; Kocer, A. Epididymis response partly compensates for spermatozoa oxidative defects in snGPx4 and GPx5 double mutant mice. PLoS One 7:e38565; 2012. [8] Arner, E. S.; Holmgren, A. Physiological functions of thioredoxin and thioredoxin reductase. Eur. J. Biochem. 267:6102–6109; 2000. [9] Miranda-Vizuete, A.; Ljung, J.; Damdimopoulos, A. E.; Gustafsson, J. A.; Oko, R.; Pelto-Huikko, M.; Spyrou, G. Characterization of Sptrx, a novel member of the thioredoxin family specifically expressed in human spermatozoa. J. Biol. Chem. 276:31567–31574; 2001. [10] Jimenez, A.; Johansson, C.; Ljung, J.; Sagemark, J.; Berndt, K. D.; Ren, B.; Tibbelin, G.; Ladenstein, R.; Kieselbach, T.; Holmgren, A.; Gustafsson, J. A.; Miranda-Vizuete, A. Human spermatid-specific thioredoxin-1 (Sptrx-1Txndc2) is a two-domain protein with oxidizing activity. FEBS Lett. 530:79–84; 2002. [11] Sadek, C. M.; Damdimopoulos, A. E.; Pelto-Huikko, M.; Gustafsson, J. A.; Spyrou, G.; Miranda-Vizuete, A. Sptrx-2, a fusion protein composed of one thioredoxin and three tandemly repeated NDP-kinase domains is expressed in human testis germ cells. Genes Cells 6:1077–1090; 2001. [12] Miranda-Vizuete, A.; Tsang, K.; Yu, Y.; Jimenez, A.; Pelto-Huikko, M.; Flickinger, C. J.; Sutovsky, P.; Oko, R. Cloning and developmental analysis of murid spermatidspecific thioredoxin-2 (SPTRX-2), a novel sperm fibrous sheath protein and autoantigen. J. Biol. Chem. 278:44874–44885; 2003. [13] Jimenez, A.; Zu, W.; Rawe, V. Y.; Pelto-Huikko, M.; Flickinger, C. J.; Sutovsky, P.; Gustafsson, J. A.; Oko, R.; Miranda-Vizuete, A. Spermatocyte/spermatid-specific thioredoxin-3, a novel Golgi apparatus-associated thioredoxin, is a specific marker of aberrant spermatogenesis. J. Biol. Chem. 279:34971–34982; 2004. [14] Jimenez, A.; Oko, R.; Gustafsson, J. A.; Spyrou, G.; Pelto-Huikko, M.; MirandaVizuete, A. Cloning, expression and characterization of mouse spermatid specific thioredoxin-1 gene and protein. Mol. Hum. Reprod. 8:710–718; 2002. [15] Biggers, J. D.; Whitten, W. K.; Whittingham, D. G. The culture of mouse embryos in vitro. In: Daniel, J. C., editor. Methods in Mammalian Embryology. San Francisco: Freeman; 1971. p. 86–116. [16] Paul, C.; Nagano, M.; Robaire, B. Aging results in differential regulation of DNA repair pathways in pachytene spermatocytes in the Brown Norway rat. Biol. Reprod 85:1269–1278; 2011. [17] Twigg, J.; Fulton, N.; Gomez, E.; Irvine, D. S.; Aitken, R. J. Analysis of the impact of intracellular reactive oxygen species generation on the structural and functional integrity of human spermatozoa: lipid peroxidation, DNA fragmentation and effectiveness of antioxidants. Hum. Reprod 13:1429–1436; 1998. [18] Koppers, A. J.; Mitchell, L. A.; Wang, P.; Lin, M.; Aitken, R. J. Phosphoinositide 3kinase signalling pathway involvement in a truncated apoptotic cascade associated with motility loss and oxidative DNA damage in human spermatozoa. Biochem. J. 436:687–698; 2011. [19] Aitken, R. J.; Whiting, S.; De Iuliis, G. N.; McClymont, S.; Mitchell, L. A.; Baker, M. A. Electrophilic aldehydes generated by sperm metabolism activate mitochondrial reactive oxygen species generation and apoptosis by targeting succinate dehydrogenase. J. Biol. Chem. 287:33048–33060; 2012. [20] Aitken, R. J.; Harkiss, D.; Knox, W.; Paterson, M.; Irvine, D. S. A novel signal transduction cascade in capacitating human spermatozoa characterised by a redox-regulated, cAMP-mediated induction of tyrosine phosphorylation. J. Cell Sci. 111:645–656; 1998. [21] Bize, I.; Santander, G.; Cabello, P.; Driscoll, D.; Sharpe, C. Hydrogen peroxide is involved in hamster sperm capacitation in vitro. Biol. Reprod. 44:398–403; 1991. [22] Aitken, R. J.; Clarkson, J. S. Cellular basis of defective sperm function and its association with the genesis of reactive oxygen species by human spermatozoa. J. Reprod. Fertil 81:459–469; 1987. [23] Aitken, R. J. The capacitation–apoptosis highway: oxysterols and mammalian sperm function. Biol. Reprod. 85:9–12; 2011. [24] Alvarez, J. G.; Touchstone, J. C.; Blasco, L.; Storey, B. T. Spontaneous lipid peroxidation and production of hydrogen peroxide and superoxide in human spermatozoa. J. Androl. 8:338–348; 1987. [25] Williams, A. C.; Ford, W. C. Functional significance of the pentose phosphate pathway and glutathione reductase in the antioxidant defenses of human sperm. Biol. Reprod 71:1309–1316; 2004. [26] Manandhar, G.; Miranda-Vizuete, A.; Pedrajas, J. R.; Krause, W. J.; Zimmerman, S.; Sutovsky, M.; Sutovsky, P. Peroxiredoxin 2 and peroxidase enzymatic activity of mammalian spermatozoa. Biol. Reprod. 80:1168–1177; 2009. [27] O'Flaherty, C.; de Souza, A. R. Hydrogen peroxide modifies human sperm peroxiredoxins in a dose-dependent manner. Biol. Reprod. 84:238–247; 2011. [28] Wood, Z. A.; Poole, L. B.; Karplus, P. A. Peroxiredoxin evolution and the regulation of hydrogen peroxide signaling. Science 300:650–653; 2003. [29] Schremmer, B.; Manevich, Y.; Feinstein, S. I.; Fisher, A. B. Peroxiredoxins in the lung with emphasis on peroxiredoxin VI. Subcell. Biochem 44:317–344; 2007. [30] Gong, S.; San Gabriel, M. C.; Zini, A.; Chan, P.; O'Flaherty, C. Low amounts and high thiol oxidation of peroxiredoxins in spermatozoa from infertile men. J. Androl. 33:1342–1351; 2012. [31] Nordberg, J.; Zhong, L.; Holmgren, A.; Arner, E. S. Mammalian thioredoxin reductase is irreversibly inhibited by dinitrohalobenzenes by alkylation of

T.B. Smith et al. / Free Radical Biology and Medicine 65 (2013) 872–881

both the redox active selenocysteine and its neighboring cysteine residue. J. Biol. Chem 273:10835–10842; 1998. [32] Schwend, T.; Moller, M.; Schabacker, J.; Ruppert, T.; Wink, M. Alkylation of adenosine deaminase and thioredoxin by acrylamide in human cell cultures. Z. Naturforsch. C 64:447–453; 2009. [33] Chen, Y.; Go, Y. M.; Pohl, J.; Reed, M.; Cai, J.; Jones, D. P. Increased mitochondrial thioredoxin 2 potentiates N-ethylmaleimide-induced cytotoxicity. Chem. Res. Toxicol. 21:1205–1210; 2008. [34] Fortin, J. S.; Cote, M. F.; Lacroix, J.; Desjardins, M.; Petitclerc, E.; C-Gaudreault, R. Selective alkylation of beta(II)-tubulin and thioredoxin-1 by structurally related subsets of aryl chloroethylureas leading to either anti-microtubules or redox modulating agents. Bioorg. Med. Chem 16:7277–7290; 2008.

881

[35] Fang, J.; Holmgren, A. Inhibition of thioredoxin and thioredoxin reductase by 4-hydroxy-2-nonenal in vitro and in vivo. J. Am. Chem. Soc 128:1879–1885; 2006. [36] De Iuliis, G. N.; Thomson, L. K.; Mitchell, L. A.; Finnie, J. M.; Koppers, A. J.; Hedges, A.; Nixon, B.; Aitken, R. J. DNA damage in human spermatozoa is highly correlated with the efficiency of chromatin remodeling and the formation of 8-hydroxy-2′-deoxyguanosine, a marker of oxidative stress. Biol. Reprod. 81:517–524; 2009. [37] Cho, C.; Jung-Ha, H.; Willis, W. D.; Goulding, E. H.; Stein, P.; Xu, Z.; Schultz, R. M.; Hecht, N. B.; Eddy, E. M. Protamine 2 deficiency leads to sperm DNA damage and embryo death in mice. Biol. Reprod. 69:211–217; 2003.