FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W4283317848> ?p ?o ?g. }

• W4283317848 endingPage "102183" @default.
• W4283317848 startingPage "102183" @default.
• W4283317848 abstract "Thioredoxin/glutathione reductase (TXNRD3) is a selenoprotein composed of thioredoxin reductase and glutaredoxin domains. This NADPH-dependent thiol oxidoreductase evolved through gene duplication within the Txnrd family, is expressed in the testes, and can reduce both thioredoxin and glutathione in vitro; however, the function of this enzyme remains unknown. To characterize the function of TXNRD3 in vivo, we generated a strain of mice bearing deletion of Txnrd3 gene. We show that these Txnrd3 knockout mice are viable and without discernable gross phenotypes, and also that TXNRD3 deficiency leads to fertility impairment in male mice. We found that Txnrd3 knockout animals exhibited a lower fertilization rate in vitro, a sperm movement phenotype, and an altered thiol redox status in sperm cells. Proteomic analyses further revealed a broad range of substrates reduced by TXNRD3 during sperm maturation, presumably as a part of sperm quality control. Taken together, these results show that TXNRD3 plays a critical role in male reproduction via the thiol redox control of spermatogenesis. Thioredoxin/glutathione reductase (TXNRD3) is a selenoprotein composed of thioredoxin reductase and glutaredoxin domains. This NADPH-dependent thiol oxidoreductase evolved through gene duplication within the Txnrd family, is expressed in the testes, and can reduce both thioredoxin and glutathione in vitro; however, the function of this enzyme remains unknown. To characterize the function of TXNRD3 in vivo, we generated a strain of mice bearing deletion of Txnrd3 gene. We show that these Txnrd3 knockout mice are viable and without discernable gross phenotypes, and also that TXNRD3 deficiency leads to fertility impairment in male mice. We found that Txnrd3 knockout animals exhibited a lower fertilization rate in vitro, a sperm movement phenotype, and an altered thiol redox status in sperm cells. Proteomic analyses further revealed a broad range of substrates reduced by TXNRD3 during sperm maturation, presumably as a part of sperm quality control. Taken together, these results show that TXNRD3 plays a critical role in male reproduction via the thiol redox control of spermatogenesis. Thioredoxin reductases (TXNRDs) are members of the pyridine nucleotide disulfide oxidoreductase family, which utilize NADPH for the reduction of their thioredoxin substrates. Three TXNRD paralogs are present in mammals: TXNRD1, TXRND2, and TXRND3 (1Sun Q.-A. Su D. Novoselov S.V. Carlson B.A. Hatfield D.L. Gladyshev V.N. Reaction mechanism and regulation of mammalian thioredoxin/glutathione reductase.Biochemistry. 2005; 44: 14528-14537Crossref PubMed Scopus (69) Google Scholar). These proteins have a C-terminal penultimate selenocysteine (Sec) residue inserted cotranslationally in response to UGA codon. TXNRD1 and TXRND2 are thioredoxin reductases in the cytosol and mitochondria, respectively, and are ubiquitously expressed in various tissues and cells (2Arner E.S.J. Focus on mammalian thioredoxin reductases — important selenoproteins with versatile functions.Biochim. Biophys. Acta. 2009; 1790: 495-526Crossref PubMed Scopus (508) Google Scholar). Previous studies revealed that TXNRD1 and TXNRD2, like their substrates (cytosolic Trx1 and mitochondrial Trx2), are essential enzymes, i.e., their knockout leads to early embryonic lethality in mice (3Jakupoglu C. Przemeck G.K.H. Schneider M. Moreno S.G. Mayr N. Hatzopoulos A.K. et al.Cytoplasmic thioredoxin reductase is essential for embryogenesis but dispensable for cardiac development.Mol. Cell. Biol. 2005; 25: 1980-1988Crossref PubMed Scopus (286) Google Scholar, 4Conrad M. Jakupoglu C. Moreno S.G. Lippl S. Banjac A. Schneider M. et al.Essential role for mitochondrial thioredoxin reductase in hematopoiesis, heart development, and heart function.Mol. Cell. Biol. 2004; 24: 9414-9423Crossref PubMed Scopus (384) Google Scholar). These and other studies revealed a critical role of TXNRDs and more generally of thiol-based redox control in mammals.The third mammalian thioredoxin reductase, TXNRD3, is unusual in that it has an additional N-terminal glutaredoxin (Grx) domain, which is fused to the canonical TXNRD module. Owing to this unusual domain organization, this enzyme can reduce both thioredoxin (Trx) and glutathione disulfide and therefore was termed thioredoxin/glutathione reductase (5Turanov A.A. Hatfield D.L. Gladyshev V.N. Characterization of protein targets of mammalian thioredoxin reductases.Methods Enzymol. 2010; 474: 245-254Crossref PubMed Scopus (7) Google Scholar). Glutathione (GSH) and Trx pathways are two major pathways that maintain the reduced state of cellular thiols and regulate redox homeostasis (6Bonilla M. Denicola A. Novoselov S.V. Turanov A.A. Protasio A. Izmendi D. et al.Platyhelminth mitochondrial and cytosolic redox homeostasis is controlled by a single thioredoxin glutathione reductase and dependent on selenium and glutathione.J. Biol. Chem. 2008; 283: 17898-17907Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), and the fact that one enzyme can support the function of both pathways is of great interest. Like other TXRNDs, TXNRD3 is a homodimer, with the monomers oriented in a head-to-tail manner. In mammals, TXNRD3 expression is largely restricted to the testis, and our previous studies suggested that TXNRD3 may be involved in the process of sperm maturation (7Su D. Novoselov S.V. Sun Q.-A. Moustafa M.E. Zhou Y. Oko R. et al.Mammalian selenoprotein thioredoxin-glutathione reductase.J. Biol. Chem. 2005; 280: 26491-26498Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). It was proposed that TXNRD3 supports disulfide bond formation and isomerization. Of interest, in platyhelminth parasites, TXNRD3 is the only TXNRD encoded in the genome, and it was found that this enzyme maintains the reduced state of GSH and Trx in both cytosol and mitochondria (6Bonilla M. Denicola A. Novoselov S.V. Turanov A.A. Protasio A. Izmendi D. et al.Platyhelminth mitochondrial and cytosolic redox homeostasis is controlled by a single thioredoxin glutathione reductase and dependent on selenium and glutathione.J. Biol. Chem. 2008; 283: 17898-17907Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar).In the current work, to dissect the role of TXNRD3 in redox homeostasis and male reproduction in vivo, we prepared mice deficient in Txnrd3. In contrast to Txnrd1 and Txnrd2 knockout mice, Txnrd3 knockout mice are viable, but the deletion of this gene leads to male fertility impairment. We show that TXNRD3 functions to support the reduction of its substrates during epididymal sperm maturation. Accordingly, TXNRD3 deficiency disrupts redox homeostasis during this process, leading to abnormal motility of the sperm and ultimately to suboptimal male fertility.ResultsEvolution of the TXNRD family to a conserved testis-specific TXNRD3In mammals, TXNRDs are selenoproteins containing a C-terminal penultimate selenocysteine (Sec), the 21st amino acid encoded by UGA. Compared with the 55-kDa subunit homodimeric TXNRD1 or TXNRD2, TXNRD3 is composed of two 65-kDa subunits because it has an additional N-terminal Grx domain (Fig. 1A). The reductase function of TXNRDs is supported by a conserved CxxxC motif, which accepts electrons from FAD thereby mediating the flow of reducing equivalents from NADPH to Trx (Fig. 1B). Our analysis (7Su D. Novoselov S.V. Sun Q.-A. Moustafa M.E. Zhou Y. Oko R. et al.Mammalian selenoprotein thioredoxin-glutathione reductase.J. Biol. Chem. 2005; 280: 26491-26498Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar) suggested that mammalian TXNRD1 and TXNRD3 evolved by gene duplication from an ancestral protein similar to fish Grx-containing TXNRD1 (Fig. 1C). TXNRD1 then lost the Grx domain (except for some rare isoforms), whereas the second Cys in the catalytic CxxC motif of the Grx domain of TNXRD3 was mutated to serine. We examined the average expression of TNXRD3 mRNA (transcripts per million) across different tissues of various species of tetrapods. Unlike TXNRD1 (present in the cytoplasm in various tissues) and TXNRD2 (present in mitochondria in various tissues), in most analyzed species TXNRD3 is highly expressed only in the testis (Fig. 1D). This observation is strongly suggestive of a role of TXNRD3 in male reproduction, as well as that this biological function is conserved across tetrapods.TXNRD3-deficient mice are viable but exhibit impaired fertilityTo get insights into the function of TXNRD3 in vivo, we generated Txnrd3 KO mice. The murine Txnrd3 gene comprises seven exons, and the Txnrd3-deficient mice were generated using a targeting strategy that replaced the third exon of Txnrd3 (Fig. S1A). ES cell clones were tested for homologous recombination by PCR and verified by Southern blotting (Fig. S1B). Upon injection into C57BL/6 blastocysts, a recombinant ES clone contributed to chimeric males, which transmitted the targeted allele to the germ line. The resulting Txnrd3-deficient mice were genotyped using mutant and allele-specific primers (Fig. S1C).This procedure yielded viable homozygous Txnrd3 KO mice, indicating that TXNRD3, in contrast to two other mammalian TXNRDs, is not essential during embryonic development and subsequent life. We also observed no other gross phenotypes in Txnrd3 KO mice, in both males and females. Since the Txnrd3 knockout allele was present in germ cells, its F1 × F1 cross should result in offspring having all three possible genotypes (Txnrd3+/+; Txnrd3+/−; Txnrd3−/−) with the Mendelian ratio of 1:2:1. However, the actual ratio was 1:1.7:0.56 (WT: heterozygous: homozygous) (Table S1). This reduction in the number of homozygous mutants suggested a possibility of fertility impairment involving Txnrd3-deficient germline. To quantify this phenotype, we set up three mating groups of mice, including WT × WT, heterozygous KO × heterozygous KO, and homozygous KO × homozygous KO, with seven pairs of animals in each group. By keeping males with females for 2 weeks and later recording the timing of females giving birth, we found that it took longer for Txnrd3-null females to give birth to offspring (WT 24.43 ± 4.54 days versus Txnrd3−/− 36.57 ± 16.27 days, p = 0.081, n = 7; WT 24.43 ± 4.54 days versus Txnrd3+/− 35.00 ± 17.18 days, p = 0.141, n = 7; Txnrd3+/− 35.00 ± 17.18 days versus Txnrd3−/− 36.57 ± 16.27 days, p = 0.863, n = 7). It is also of note that two Txnrd3−/− females did not give birth (they were monitored for 60 days) (Fig. 2A), supporting a potential role of TXNRD3 for the germline. In addition, there were fewer pups per litter in the case of homozygous KO mice (WT 7.43 ± 1.72 versus Txnrd3−/− 3.71 ± 2.69, p = 0.010, n = 7; WT 7.43 ± 1.72 versus Txnrd3+/− 4.29 ± 2.98, p = 0.033, n = 7; Txnrd3+/− 4.29 ± 2.98 versus Txnrd3−/− 3.71 ± 2.69, p = 0.713, n = 7) (Fig. 2B).Figure 2Deletion of mouse Txnrd3 gene impairs male fertility. A, period from the beginning of mating to females giving birth. Two females in the heterozygous and homozygous groups did not give birth for 60 days after the beginning of mating. WT, wildtype group. B, litter size in each animal group. C, number of cauda sperm in WT and Txnrd3-null male mice. D, representative image of fertilized embryos from the cauda sperm from WT (left) and Txnrd3-null (right) males in vitro. E, fertilization rates of WT and Txnrd3-null animals in vitro.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We further examined sperm counts of Txnrd3-null and WT mice and found that the number of spermatozoa in Txnrd3-null mice was significantly lower than in WT mice (WT 2.96 ± 0.64 × 107 versus Txnrd3−/− 2.26 ± 0.55 × 107, p = 0.048, n = 7) (Fig. 2C). To further explore fertility of Txnrd3-null mice, we carried out in vitro fertilization. The in vitro fertilization rate with cumulus oocyte complex-intact eggs was significantly lower when the sperm from Txnrd3−/− males was compared with the WT sperm (WT 43.40 ± 8.15 versus Txrnd3−/− 29.03 ± 1.78, p = 0.041, n = 3) (Fig. 2, D and E). Finally, we examined movement of free-swimming spermatozoa isolated from WT, Txnrd3+/−, and Txnrd3−/− mice. Compared with WT mice, the fraction of motile spermatozoa was visibly lower in Txnrd3−/− mice (Movie S1).Taken together, our data suggest that, although TXNRD3 is neither essential for development nor for male fertility in general, its KO leads to reduced sperm motility and fertility, making these male subfertile. In the accompanying article (8Wang H. Dou Q. Jung K.J. Choi J. Gladyshev V.N. Chung J.-J. Redox regulation by TXNRD3 during epididymal maturation underlies capacitation-associated mitochondrial activation and sperm motility in mice.J. Biol. Chem. 2021; 298: 102077Abstract Full Text Full Text PDF Scopus (3) Google Scholar), we provide further evidence for the role of TXNRD3 in epididymal maturation, and specifically its impact on capacitation-associated mitochondrial activation and sperm motility.Thiol peroxidases as interacting partners of TXNRD3Previous functional characterization and localization of TXNRD3 revealed its possible disulfide bond isomerization activity and a close functional relationship to another selenoprotein, GPX4 (7Su D. Novoselov S.V. Sun Q.-A. Moustafa M.E. Zhou Y. Oko R. et al.Mammalian selenoprotein thioredoxin-glutathione reductase.J. Biol. Chem. 2005; 280: 26491-26498Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). The use of String to examine the association between TXNRD3 and GPX4 showed a combined score of 0.717, suggesting a high confidence functional link between these two proteins (Fig. S2A). To further characterize this association, we analyzed protein levels of TXNRD3 and GPX4 in the mouse testis and sperm by immunoblotting. Interesting, GPX4 expression was higher in Txnrd3−/− testes than in WT testes (WT 0.77 ± 0.11 versus Txnrd3−/− 0.96 ± 0.04, p = 0.049, n = 3; WT 0.77 ± 0.11 versus Txnrd3+/− 0.82 ± 0.02, p = 0.424, n = 3; Txnrd3+/− 0.82 ± 0.02 versus Txnrd3−/− 0.96 ± 0.04, p = 0.006, n = 3) (Fig. 3A), whereas the expression of this protein in the sperm was not much affected (Fig. 3B). Immunohistochemical staining of TXNRD3 and GPX4 showed a similar pattern (Fig. 3, C and D).Figure 3Expression of TXNRD3 and GPX4 in mouse testis and sperm. A, TXNRD3 and GPX4 expression in testis based on Western blotting. The right panel quantifies GPX4. Liver sample was used for comparison with the testis. Because the expression of GPX4 is much higher in the testis than in the liver, it appears as not detectable. B, TXNRD3 and GPX4 levels in mouse sperm. The right panel quantifies GPX4. C, immunohistochemistry of GPX4 in Txnrd3 KO and WT mouse testis and sperm (caput and cauda). D, immunohistochemistry of TXNRD3 in Txnrd3 KO and WT mouse testes and sperm (caput and cauda).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Another group of thiol peroxidases implicated in male reproduction are peroxiredoxins (PRDXs) (9Ryu D.-Y. Kim K.-U. Kwon W.-S. Rahman M.S. Khatun A. Pang M.-G. Peroxiredoxin activity is a major landmark of male fertility.Sci. Rep. 2017; 7: 17174Crossref PubMed Scopus (27) Google Scholar). In particular, peroxiredoxin 4 (PRDX4) is thought to play a role in this process by regulating oxidative packaging of sperm chromatin (10Fujii J. Ikeda Y. Kurahashi T. Homma T. Physiological and pathological views of peroxiredoxin 4.Free Radic. Biol. Med. 2015; 83: 373-379Crossref PubMed Scopus (37) Google Scholar). Based on String, the association among PRDX4, GPX4, and TXNRD3 showed a medium confidence (Fig. S2A). We detected the expression of PRDX4 in mouse testis and sperm, and its expression in the testis was particularly high. We also found that PRDX4 expression was decreased in Txnrd3-deficient testis compared with WT (Fig. 2, B and C). Altogether, these analyses were suggestive of a possible functional association of TXNRD3 with thiol peroxidases of the GPX and PRDX families, but further studies are needed to clarify this possibility.A role of TXNRD3 in maintaining thiol redox status of sperm proteinsRedox homeostasis has been identified as a critical factor in male infertility (11Agarwal A. Virk G. Ong C. du Plessis S.S. Effect of oxidative stress on male reproduction.World J. Mens Health. 2014; 32: 1-17Crossref PubMed Google Scholar), as spermatogenesis is associated with dramatic redox transitions leading to widespread oxidation of thiols. Consistent with this notion, sperm is particularly susceptible to reactive oxygen species (ROS) during critical phases of sperm development (12Sabeti P. Pourmasumi S. Rahiminia T. Akyash F. Talebi A.R. Etiologies of sperm oxidative stress.Int. J. Reprod. Biomed. 2016; 14: 231-240PubMed Google Scholar). Based on our immunohistochemical staining (Fig. 3C) and Fig. S2 in our companion article (8Wang H. Dou Q. Jung K.J. Choi J. Gladyshev V.N. Chung J.-J. Redox regulation by TXNRD3 during epididymal maturation underlies capacitation-associated mitochondrial activation and sperm motility in mice.J. Biol. Chem. 2021; 298: 102077Abstract Full Text Full Text PDF Scopus (3) Google Scholar), TXNRD3 is highly expressed in spermatogonia, spermatocytes, and early spermatids, so its deficiency may be particularly detrimental for the thiol redox transition. By using CM-H2DCFDA to detect ROS, we found no significant differences between KO and WT sperm (Fig. 4A). This is not unexpected—while intracellular thiols are among the major targets of ROS (13Baba S.P. Bhatnagar A. Role of thiols in oxidative stress.Curr. Opin. Toxicol. 2018; 7: 133-139Crossref PubMed Scopus (77) Google Scholar), thiol redox dysfunction does not necessarily have to involve ROS. To assess potential protein thiol changes in the Txnrd3 KO sperm, we employed BODIPY-NEM as a fluorescent probe to directly label thiols in sperm samples (14Hill B.G. Reily C. Oh J.-Y. Johnson M.S. Landar A. Methods for the determination and quantification of the reactive thiol proteome.Free Radic. Biol. Med. 2009; 47: 675-683Crossref PubMed Scopus (75) Google Scholar), followed by flow cytometry. We observed a lower percentage of BODIPY-NEM-positive sperm in Txnrd3−/− than in WT mice (WT 86.57 ± 6.12 versus Txnrd3−/− 64.17 ± 9.12, p = 0.024, n = 3) (Fig. 4B), consistent with the idea that TXNRD3 supports the formation or maintenance of thiols during sperm maturation.Figure 4Analyses of redox characteristics of sperm. A, reactive oxygen species levels in mouse sperm as revealed by CM-H2DCFDA assays. B, protein thiols in mouse sperm as revealed by using BODIPY-NEM. C, expression of Trx1, GSTa2, and GSTm1 in mouse testis and sperm by Western blotting.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The most abundant thiol in animal cells is glutathione (GSH), which is present both in the cytosol and various organelles (15Wu G. Fang Y.-Z. Yang S. Lupton J.R. Turner N.D. Glutathione metabolism and its implications for Health.J. Nutr. 2004; 134: 489-492Crossref PubMed Scopus (2562) Google Scholar). We examined the levels of glutathionylated proteins and found no differences between WT, Txnrd3+/−, and Txnrd3−/− mice (Fig. 5A). In addition, we performed immunoprecipitation to enrich glutathionylated proteins in mouse testis and sperm and again found no differences among animal groups (Fig. 5, B and C). We also tested thioredoxin 1 (Trx1), glutathione S-transferase A2 (GSTa2), and glutathione S-transferase Mu 1 (GSTm1) expression in the testis and sperm (Fig. 4C). The expression of Trx1 in Txnrd3−/− sperm was elevated. This is a conserved thiol oxidoreductase that maintains protein thiols in the reduced state; it is also a substrate for TXRNDs, including TXNRD3 (16Wu C. Parrott A.M. Fu C. Liu T. Marino S.M. Gladyshev V.N. et al.Thioredoxin 1-mediated post-translational modifications: reduction, transnitrosylation, denitrosylation, and related proteomics methodologies.Antioxid. Redox Signal. 2011; 15: 2565-2604Crossref PubMed Scopus (86) Google Scholar). The increased Trx1 expression in Txnrd3−/− sperm and testes might be a compensatory mechanism in view of TXNRD3 deficiency.Figure 5Analyses of glutathionylation in mouse testis and sperm. A, glutathionylated proteins in the testis of WT and Txnrd3 KO mice (and liver as control) as analyzed by Western blotting with anti-GSH antibodies. B, immunoprecipitation (IP) of glutathionylated proteins in WT and Txnrd3 KO testes with anti-GSH antibodies. Initial (corresponds to whole cell lysate), flow through (flow), and elution (elution) factions were analyzed by Western blotting using anti-GSH and TXNRD3 antibodies (left panel). Protein staining with Amido Black (right panel). C, immunoprecipitation (IP) of glutathionylated proteins in WT mice and Txnrd3 KO sperm with anti-GSH antibodies. Initial (corresponding to whole cell lysate), flow through (flow), and elution (elution) factions from a GSH enrichment experiment were analyzed by Western blotting using anti-GSH and TXNRD3 antibodies (left panel). Protein staining with Amido Black (right panel).View Large Image Figure ViewerDownload Hi-res image Download (PPT)During spermatogenesis, the sperm DNA becomes highly compact. To facilitate this extreme transition, the vast majority of somatic histones are replaced by small basic proteins, and then these proteins undergo thiol oxidation to form intramolecular and intermolecular disulfide bonds (17Miller D. Brinkworth M. Iles D. Paternal DNA packaging in spermatozoa: more than the sum of its parts? DNA, histones, protamines and epigenetics.Reproduction. 2010; 139: 287-301Crossref PubMed Scopus (285) Google Scholar). These disulfides are crucial for DNA packaging, as its mispackaging in the sperm head results in decreased fertility, higher rates of miscarriage, and higher rates of genetic disease in the offspring (18Hutchison J.M. Rau D.C. DeRouchey J.E. Role of disulfide bonds on DNA packaging forces in bull sperm chromatin.Biophys. J. 2017; 113: 1925-1933Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). To test the possibility that TXNRD3 deficiency affects disulfide bond formation, we employed diagonal gel electrophoresis, thereby examining oligomeric proteins containing interchain disulfide bonds. Overall protein patterns of both WT and Txnrd3−/− testes and sperm showed similar patterns of protein cross-linking (Fig. S3). Our findings suggest dysfunction in thiol redox homeostasis might occur during spermatogenesis and sperm maturation in Txnrd3−/− males.Putative TXNRD3 targetsAs TXNRD3 exhibits NADPH-dependent thiol reductase activities in vitro, and its deficiency reduces the levels of thiols in sperm, a reasonable possibility is that this enzyme supports the reduction of disulfide-containing substrates during sperm maturation. Aiming to detect such putative endogenous TXNRD3 targets, we adapted a procedure for their enrichment and applied it to three different areas of the epididymis (caput, corpus, and cauda), profiling the epididymal luminal content of Txnrd3 KO and WT sperm (eight mice per group). This approach was based on the assumption that misoxidized target proteins are normally reduced by TXNRD3 in WT mice and therefore may proceed to their correct oxidized state during sperm maturation, whereas these proteins partially remain in the incorrect oxidized form in Txnrd3 KO mice (Fig. S4). As a first step, free thiols in sperm lysates of Txnrd3 KO and WT mice were blocked with N-ethyl maleimide (NEM), and the lysates were applied to a column containing the Grx domain of TXNRD3, which was in the pre-reduced state (corresponding to the NADPH-reduced state of TXNRD3). The targets reduced by this domain were then applied to a thiol-trapping column and eluted with DTT. The resulting eluates, putatively enriched for TXNRD3 targets, were treated with iodoacetamide to block any free thiols and subjected to proteomics analyses. Spectral counts of detected proteins (Fig. S5 and Table S2) were normalized using the Normalized Spectral Abundance Factor approach (19Paoletti A.C. Parmely T.J. Tomomori-Sato C. Sato S. Zhu D. Conaway R.C. et al.Quantitative proteomic analysis of distinct mammalian mediator complexes using normalized spectral abundance factors.Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 18928-18933Crossref PubMed Scopus (401) Google Scholar, 20Zybailov B.L. Florens L. Washburn M.P. Quantitative shotgun proteomics using a protease with broad specificity and normalized spectral abundance factors.Mol. Biosyst. 2007; 3: 354-360Crossref PubMed Scopus (117) Google Scholar). Interestingly, we detected more proteins in KO than in WT samples, consistent with the broad range of substrates. To uncover relevant patterns of proteins present in Txnrd3 KO but not in WT samples, we devised four selection criteria (Fig. S6), and their application to the proteomic dataset led to a set of putative target proteins (Fig. 6). Interestingly, this approach did not reveal a single bona fide TXNRD3 target. Instead, we detected a broad range of target proteins. Based on functional enrichment analysis (DAVID functional annotation tool v6.8), the set of putative targets (103 proteins) was enriched for proteins annotated as RNA binding (p-value = 1.77E-5; false discovery rate = 0.024). In addition, a set of mitochondrial proteins (ten proteins) involved in metabolism were detected only in the cauda sperm from Txnrd3 KO mice, suggesting that TXNRD3 deficiency might affect ATP production by sperm cells. This finding is consistent with the idea that TXNRD3 reduces a broad set of proteins during spermatogenesis and sperm maturation.Figure 6Putative TXNRD3 target proteins. Heatmap shows absolute peptide counts per protein. The left dendrogram represents a clustering structure of candidate proteins (obtained by the R function hclust, ward.D method, applied to the Euclidean distance matrix). On the right of the heatmap, colored shapes show, for each protein, to which candidate set(s) it belongs to (Fig. S4). Finally, the name of candidate proteins is reported on the right.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DiscussionBoth previously characterized mammalian TXNRD family members, i.e., cytosolic TXNRD1 and mitochondrial TXNRD2, are essential proteins. As the third member of this family, TXNRD3, is abundantly expressed in the testes across tetrapods, we considered a possibility that this protein instead plays an essential role in spermatogenesis. We developed Txnrd3 KO mice and, as predicted, found that they were viable and without gross phenotypes. Unexpectedly, however, Txnrd3 KO males were fertile, i.e., TXNRD3 was not essential for male reproduction. However, by applying a mating scheme to more carefully examine fertility of WT and KO animals, we found that fertility of Txnrd3−/− males was in fact partially compromised. In addition, an in vitro fertilization experiment showed a lower fertilization rate of Txnrd3−/− mice compared with WT mice, and the Txnrd3−/− sperm exhibited an altered state of thiols. These data revealed that, although TXNRD3 is not essential for male reproduction, it plays an important role in this process by supporting redox homeostasis during spermatogenesis. As TXNRD3 is conserved in mammals, presumably its function is also conserved, and the subfertility associated with its deficiency must be severe enough in the wild to preserve the protein in these organisms over great evolutionary distances.Owing to is domain organization, TXNRD3 may be viewed as a component of both Trx and GSH systems. The Trx system provides reducing equivalents to many proteins, and especially to thiol peroxidases, such as PRDXs. It was found that Trx1 can be directly reduced by TXNRD3 and TXNRD3 could support the reduction of thiol peroxidases via thioredoxin (21Lu J. Holmgren A. The thioredoxin antioxidant system.Free Radic. Biol. Med. 2014; 66: 75-87Crossref PubMed Scopus (1086) Google Scholar, 22Dorey A. Cwiklinski K. Rooney J. De Marco Verissimo C. López Corrales J. Jewhurst H. et al.Autonomous non antioxidant roles for fasciola hepatica secreted thioredoxin-1 and peroxiredoxin-1.Front. Cell. Infect. Microbiol. 2021; 11: 667272Crossref PubMed Scopus (7) Google Scholar). In addition, Trx1 expression was higher in Txnrd3−/− mice sperm (compared with WT mice), suggesting a possible compensatory mechanism. It would be interesting to examine whether the elevated Trx1 is also altered with regard to its redox state. An additional compensatory mechanism could be provided by the elevated expression of GSTs, in combination with an altered level of redox state of glutathione. An attractive possibility is that such mechanisms partially rescue TXNRD3 function, precluding male infertility.One of PRDXs, PRDX4, was also implicated in male reproduction through redox homeostasis (23Tasaki E. Matsumoto S. Tada H. Kurahashi T. Zhang X. Fujii J. et al.Protective role of testis-specific peroxiredoxin 4 against cellular oxidative stress.J. Clin. Biochem. Nutr. 2017; 60: 156-161Crossref PubMed Scopus (3) Google Scholar). An additional thiol peroxidase, which is a k" @default.
• W4283317848 created "2022-06-24" @default.
• W4283317848 creator A5004540478 @default.
• W4283317848 creator A5005451056 @default.
• W4283317848 creator A5010080171 @default.
• W4283317848 creator A5014486696 @default.
• W4283317848 creator A5015505425 @default.
• W4283317848 creator A5023061926 @default.
• W4283317848 creator A5026734370 @default.
• W4283317848 creator A5039607842 @default.
• W4283317848 creator A5047225117 @default.
• W4283317848 creator A5052321093 @default.
• W4283317848 creator A5082613226 @default.
• W4283317848 date "2022-08-01" @default.
• W4283317848 modified "2023-10-14" @default.
• W4283317848 title "Selenoprotein TXNRD3 supports male fertility via the redox regulation of spermatogenesis" @default.
• W4283317848 cites W1533942137 @default.
• W4283317848 cites W1574664331 @default.
• W4283317848 cites W1809645018 @default.
• W4283317848 cites W1891967301 @default.
• W4283317848 cites W1963294633 @default.
• W4283317848 cites W1963783185 @default.
• W4283317848 cites W1965277334 @default.
• W4283317848 cites W1966815619 @default.
• W4283317848 cites W1969885044 @default.
• W4283317848 cites W1978772332 @default.
• W4283317848 cites W1984140215 @default.
• W4283317848 cites W1984568177 @default.
• W4283317848 cites W1989800916 @default.
• W4283317848 cites W2006866503 @default.
• W4283317848 cites W2012455936 @default.
• W4283317848 cites W2018745521 @default.
• W4283317848 cites W2025619959 @default.
• W4283317848 cites W2027249101 @default.
• W4283317848 cites W2032601646 @default.
• W4283317848 cites W2047437538 @default.
• W4283317848 cites W2068034831 @default.
• W4283317848 cites W2070472037 @default.
• W4283317848 cites W2086278931 @default.
• W4283317848 cites W2092756750 @default.
• W4283317848 cites W2103336074 @default.
• W4283317848 cites W2104535491 @default.
• W4283317848 cites W2115560639 @default.
• W4283317848 cites W2116487802 @default.
• W4283317848 cites W2116948221 @default.
• W4283317848 cites W2171813756 @default.
• W4283317848 cites W2172183886 @default.
• W4283317848 cites W2223764567 @default.
• W4283317848 cites W2225176222 @default.
• W4283317848 cites W2330166382 @default.
• W4283317848 cites W2337978407 @default.
• W4283317848 cites W2342500436 @default.
• W4283317848 cites W2403177680 @default.
• W4283317848 cites W2577191321 @default.
• W4283317848 cites W2604562271 @default.
• W4283317848 cites W2609355423 @default.
• W4283317848 cites W2749117395 @default.
• W4283317848 cites W2767789945 @default.
• W4283317848 cites W2775187721 @default.
• W4283317848 cites W2778087102 @default.
• W4283317848 cites W2802092176 @default.
• W4283317848 cites W2899971546 @default.
• W4283317848 cites W2942504068 @default.
• W4283317848 cites W3094200629 @default.
• W4283317848 cites W3120660874 @default.
• W4283317848 cites W3143723535 @default.
• W4283317848 cites W3159585578 @default.
• W4283317848 cites W4281568460 @default.
• W4283317848 doi "https://doi.org/10.1016/j.jbc.2022.102183" @default.
• W4283317848 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/35753352" @default.
• W4283317848 hasPublicationYear "2022" @default.
• W4283317848 type Work @default.
• W4283317848 citedByCount "8" @default.
• W4283317848 countsByYear W42833178482022 @default.
• W4283317848 countsByYear W42833178482023 @default.
• W4283317848 crossrefType "journal-article" @default.
• W4283317848 hasAuthorship W4283317848A5004540478 @default.
• W4283317848 hasAuthorship W4283317848A5005451056 @default.
• W4283317848 hasAuthorship W4283317848A5010080171 @default.
• W4283317848 hasAuthorship W4283317848A5014486696 @default.
• W4283317848 hasAuthorship W4283317848A5015505425 @default.
• W4283317848 hasAuthorship W4283317848A5023061926 @default.
• W4283317848 hasAuthorship W4283317848A5026734370 @default.
• W4283317848 hasAuthorship W4283317848A5039607842 @default.
• W4283317848 hasAuthorship W4283317848A5047225117 @default.
• W4283317848 hasAuthorship W4283317848A5052321093 @default.
• W4283317848 hasAuthorship W4283317848A5082613226 @default.
• W4283317848 hasBestOaLocation W42833178481 @default.
• W4283317848 hasConcept C123765429 @default.
• W4283317848 hasConcept C134018914 @default.
• W4283317848 hasConcept C16685009 @default.
• W4283317848 hasConcept C178790620 @default.
• W4283317848 hasConcept C185592680 @default.
• W4283317848 hasConcept C2775838275 @default.
• W4283317848 hasConcept C2776151105 @default.
• W4283317848 hasConcept C2776841403 @default.
• W4283317848 hasConcept C2778760513 @default.
• W4283317848 hasConcept C2908647359 @default.

• next