Peroxiredoxin activity is a major landmark of male fertility.
ABSTRACT: Peroxiredoxins (PRDXs) are important antioxidant enzymes reported to have a role in sperm function and male fertility. However, how PRDXs affects male fertility remain fundamental unanswered questions. We therefore sought to investigate the role of these enzymes in sperm function and fertilisation. In this in vitro trial, mouse spermatozoa were incubated with different concentrations of conoidin A (1, 10, or 100?µM), a specific inhibitor of PRDXs. Our results demonstrated that inhibition of PRDXs by conoidin A significantly decreased the oxidized form of peroxiredoxins (PRDXs-SO3) in spermatozoa. Decreased PRDX activity was associated with a significant reduction in sperm motility parameters, viability, and intracellular ATP, whereas ROS levels, DNA fragmentation, and loss of mitochondrial membrane potential were increased. Simultaneously capacitation and the acrosome reaction were also significantly inhibited perhaps as a consequence of decreased tyrosine phosphorylation and protein kinase-A activity. In addition, fertilisation and early embryonic development were adversely affected following PRDXs inhibition in spermatozoa. Taken together, our data demonstrate that decreased PRDX activity directly affects male fertility due to negative effects on important functions and biochemical properties of spermatozoa, ultimately leading to poor fertilisation and embryonic development.
Project description:STUDY QUESTION:Are all components of the peroxiredoxins (PRDXs) system important to control the levels of reactive oxygen species (ROS) to maintain viability and DNA integrity in spermatozoa? SUMMARY ANSWER:PRDX6 is the primary player of the PRDXs system for maintaining viability and DNA integrity in human spermatozoa. WHAT IS KNOWN ALREADY:Mammalian spermatozoa are sensitive to high levels of ROS and PRDXs are antioxidant enzymes proven to control the levels of ROS generated during sperm capacitation to avoid oxidative damage in the spermatozoon. Low amounts of PRDXs are associated with male infertility. The absence of PRDX6 promotes sperm oxidative damage and infertility in mice. STUDY DESIGN, SIZE, DURATION:Semen samples were obtained over a period of one year from a cohort of 20 healthy non-smoking volunteers aged 22-30 years old. PARTICIPANTS/MATERIALS, SETTING, METHODS:Sperm from healthy donors was incubated for 2 h in the absence or presence of inhibitors for the 2-Cys PRDXs system (peroxidase, reactivation system and NADPH-enzymes suppliers) or the 1-Cys PRDX system (peroxidase and calcium independent-phospholipase A2 (Ca2+-iPLA2) activity). Sperm viability, DNA oxidation, ROS levels, mitochondrial membrane potential and 4-hydroxynonenal production were determined by flow cytometry. MAIN RESULTS AND THE ROLE OF CHANCE:We observed a significant decrease in viable cells due to inhibitors of the 2-Cys PRDXs, PRDX6 Ca2+-iPLA2 activity or the PRDX reactivation system compared to controls (P ? 0.05). PRDX6 Ca2+-iPLA2 activity inhibition had the strongest detrimental effect on sperm viability and DNA oxidation compared to controls (P ? 0.05). The 2-Cys PRDXs did not compensate for the inhibition of PRDX6 peroxidase and Ca2+-iPLA2 activities. LARGE SCALE DATA:Not applicable. LIMITATIONS, REASONS FOR CAUTION:Players of the reactivation systems may differ among mammalian species. WIDER IMPLICATIONS OF THE FINDINGS:The Ca2+-iPLA2 activity of PRDX6 is the most important and first line of defense against oxidative stress in human spermatozoa. Peroxynitrite is scavenged mainly by the PRDX6 peroxidase activity. These findings can help to design new diagnostic tools and therapies for male infertility. STUDY FUNDING/COMPETING INTEREST(S):This research was supported by The Canadian Institutes of Health Research (MOP 133661 to C.O.), and by RI MUHC-Desjardins Studentship in Child Health Research awarded to M.C.F. The authors have nothing to disclose.
Project description:Peroxiredoxins (PRDXs) are antioxidant enzymes that protect cells from oxidative stress and play a role in reactive oxygen species (ROS)-mediated signaling. We reported that PRDXs are critical for human fertility by maintaining sperm viability and regulating ROS levels during capacitation. Moreover, studies on Prdx6-/- mice revealed the essential role of PRDX6 in the viability, motility, and fertility competence of spermatozoa. Although PRDXs are abundant in the testis and spermatozoa, their potential role at different phases of spermatogenesis and in perinatal germ cells is unknown. Here, we examined the expression and role of PRDXs in isolated rat neonatal gonocytes, the precursors of spermatogonia, including spermatogonial stem cells. Gene array, qPCR analyses showed that PRDX1, 2, 3, 5, and 6 transcripts are among the most abundant antioxidant genes in postnatal day (PND) 3 gonocytes, while immunofluorescence confirmed the expression of PRDX1, 2, and 6 proteins. The role of PRDXs in gonocyte viability was examined using PRDX inhibitors, revealing that the 2-Cys PRDXs and PRDX6 peroxidases activities are critical for gonocytes viability in basal condition, likely preventing an excessive accumulation of endogenous ROS in the cells. In contrast to its crucial role in spermatozoa, PRDX6 independent phospholipase A2 (iPLA2) activity was not critical in gonocytes in basal conditions. However, under conditions of H2O2-induced oxidative stress, all these enzymatic activities were critical to maintain gonocyte viability. The inhibition of PRDXs promoted a two-fold increase in lipid peroxidation and prevented gonocyte differentiation. These results suggest that ROS are produced in neonatal gonocytes, where they are maintained by PRDXs at levels that are non-toxic and permissive for cell differentiation. These findings show that PRDXs play a major role in the antioxidant machinery of gonocytes, to maintain cell viability and allow for differentiation.
Project description:Previous studies have demonstrated that thioredoxin 1 (Trx1) exerts neuroprotective effects against cerebral ischemia/reperfusion injury caused by oxidative stress. While Trx1 is known to maintain the anti?oxidant activity of 2?Cys peroxiredoxins (Prdxs), the underlying mechanisms of its protective effects have remained to be elucidated, which was the aim of the present study. For this, an in vitro ischemic model of hypoxemia lasting for 4 h, followed by 24 h of reperfusion was used. Primary astrocytes from neonatal rats were pre?treated with small interfering RNA targeting Trx1 prior to oxygen glucose deprivation/reperfusion (OGD/R). MTS and lactate dehydrogenase assays were performed to evaluate cell viability. Reverse transcription?quantitative polymerase chain reaction (RT?qPCR) and western blot analysis were employed to assess the mRNA and protein expression levels of Prdx1?4 and Prdx?SO3. Furthermore, a dual luciferase reporter assay was used to assess the interaction between activator protein?1 (AP?1) and Trx1. The present study demonstrated that OGD/R decreased the cell viability and increased cellular damage, which was more marked following Trx1 knockdown. The expression of Prdx1?4 and Prdx?SO3 protein was higher in the cells subjected to OGD/R. Knockdown of Trx1 markedly decreased the levels of Prdx1?4 but increased Prdx?SO3 mRNA and protein levels. The results of the present study also suggested that AP?1 directly activated the expression of Trx1. The present study demonstrated that Trx1 exerts its neuroprotective effects by preventing oxidative stress in astrocytes via maintaining Prdx expression.
Project description:Oxidative stress is a common culprit of several conditions associated with male fertility. High levels of reactive oxygen species (ROS) promote impairment of sperm quality mainly by decreasing motility and increasing the levels of DNA oxidation. Oxidative stress is a common feature of environmental pollutants, chemotherapy and other chemicals, smoke, toxins, radiation, and diseases that can have negative effects on fertility. Peroxiredoxins (PRDXs) are antioxidant enzymes associated with the protection of mammalian spermatozoa against oxidative stress and the regulation of sperm viability and capacitation. In the present study, we aimed to determine the long-term effects of oxidative stress in the testis, epididymis and spermatozoa using the rat model. Adult male rats were treated with tert-butyl hydroperoxide (t-BHP) or saline (control group), and reproductive organs and spermatozoa were collected at 3, 6, and 9 weeks after the end of treatment. We determined sperm DNA oxidation and motility, and levels of lipid peroxidation and protein expression of antioxidant enzymes in epididymis and testis. We observed that cauda epididymal spermatozoa displayed low motility and high DNA oxidation levels at all times. Lipid peroxidation was higher in caput and cauda epididymis of treated rats at 3 and 6 weeks but was similar to control levels at 9 weeks. PRDX6 was upregulated in the epididymis due to t-BHP; PRDX1 and catalase, although not significant, followed similar trend of increase. Testis of treated rats did not show signs of oxidative stress nor upregulation of antioxidant enzymes. We concluded that t-BHP-dependent oxidative stress promoted long-term changes in the epididymis and maturing spermatozoa that result in the impairment of sperm quality.
Project description:Recent studies have demonstrated the significance of sperm RNA function as a transporter of important information directing the course of life. To determine the message contained in sperm RNA, it is necessary to optimize transcriptomic research tools. The current study was performed to optimize the processing of sperm RNA from sample storage to quantitative real-time PCR and assess the corresponding method with to evaluate male fertility and its representative markers, equatorin (EQTN) and peroxiredoxin (PRDX). Following successive steps of the Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines, several options were compared using boar spermatozoa. To evaluate the optimized procedures, the relationship between mRNA expression of EQTN and PRDX in spermatozoa and the fertility (litter size) of 20 individual boars was assessed. Unexpectedly, DNase treatment during RNA isolation had the deleterious effect by decreasing the RNA concentration by 56% and eliminating the correlation between EQTN and PRDX4 mRNA expression and male fertility. Moreover, when sperm RNA was processed using the corresponding method, the results showed the highest exon sequence expression, male fertility prediction power, and consistency. This optimized protocol for predicting male fertility can be used to study the transport of messages directing the life course from spermatozoon to offspring.
Project description:Do peroxiredoxins (PRDXs) control reactive oxygen species (ROS) levels during human sperm capacitation?PRDXs are necessary to control the levels of ROS generated during capacitation allowing spermatozoa to achieve fertilizing ability.Sperm capacitation is an oxidative event that requires low and controlled amounts of ROS to trigger phosphorylation events. PRDXs are antioxidant enzymes that not only act as scavengers but also control ROS action in somatic cells. Spermatozoa from infertile men have lower levels of PRDXs (particularly of PRDX6), which are thiol-oxidized and therefore inactive.Semen samples were obtained from a cohort of 20 healthy nonsmoker volunteers aged 22-30 years old over a period of 1 year.Sperm from healthy donors was capacitated with fetal cord serum ultrafiltrate (FCSu) in the absence or presence of thiostrepton (TSP), inhibitor of 2-Cys PRDXs or 1-Hexadecyl-3-(trifluoroethyl)-sn-glycero-2-phosphomethanol lithium (MJ33), inhibitor of calcium independent-phospholipase A2 (Ca2+-iPLA2) activity of PRDX6, added at different times of incubation. Capacitation was also induced by the dibutyryl cAMP+3-isobuty1-1-methylxanthine system. Sperm viability and motility were determined by the hypo-osmotic swelling test and computer-assisted semen analysis system, respectively. Capacitation was determined by the ability of spermatozoa to undergo the acrosome reaction triggered by lysophosphatidylcholine. Percentages of acrosome reaction were obtained using the FITC-conjugated Pisum sativum agglutinin assay. Phosphorylation of tyrosine residues and of protein kinase A (PKA) substrates were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis immunoblotting with specific antibodies. Actin polymerization was determined by phalloidin labeling.TSP and MJ33 prevented sperm capacitation and its associated actin polymerization in spermatozoa incubated with 10% FCSu (capacitation inducer) compared to non-capacitated controls (P < 0.05) without altering sperm viability. PKA substrates and tyrosine phosphorylations were prevented in FCSu-treated spermatozoa in a differential fashion depending on the type and the time of addition of the inhibitor used compared to non-capacitated controls (P < 0.05). TSP and MJ33 promoted an increase of lipid peroxidation in spermatozoa (P < 0.01) and these levels were higher in those spermatozoa incubated with the inhibitors and FCSu compared to those capacitated spermatozoa incubated without the inhibitors (P < 0.0001). Inhibition of 2-Cys PRDXs by TSP generated an oxidative stress in spermatozoa, affecting their viability compared to controls (P < 0.05). This oxidative stress was prevented by nuclephile D-penicillamine (PEN). MJ33 also promoted an increase of lipid peroxidation and impaired sperm viability compared to non-treated controls (P < 0.05) but its effect was not circumvented by PEN, suggesting that not only peroxidase but also Ca2+-iPLA2 activity of PRDX6 are necessary to guarantee viability in human spermatozoa.Not applicable.We focused on the global effect of PRDXs inhibitors on human sperm capacitation and in two of its associated phosphorylation events. Thus, other phosphorylation events and mechanisms necessary for capacitation may also be affected.PRDXs are the major antioxidant system in ejaculated spermatozoa and are necessary to allow spermatozoon to achieve fertilizing ability (capacitation and acrosome reaction).This research was supported by Canadian Institutes of Health Research (MOP 133661) and the Fonds de Recherché en Santé Quebec (FRSQS #22151) to C.O. The authors have nothing to disclose.
Project description:Prdx6 -/- male mice are subfertile, and the deficiency or inactivation of Peroxiredoxins (PRDXs) is associated with human male infertility. We elucidate the impact of the lack of PRDX6 or inhibition of its calcium-independent phospholipase A2 (Ca2+-iPLA2) activity by MJ33 on fertilization competence of mouse spermatozoa. Sperm motility, viability, fertilization and blastocyst rates were lower in Prdx6 -/- spermatozoa than in C57BL/6J wild-type (WT) controls (p???0.05). MJ33 inhibited the PRDX6 Ca2+-iPLA2 activity and reduced these parameters in WT spermatozoa compared with controls (p???0.05). Levels of lipid peroxidation and of superoxide anion (O2•?) were higher in Prdx6 -/- than in WT spermatozoa (p???0.05). MJ33 increased the levels of lipid peroxidation and mitochondrial O2•? production in treated versus non-treated WT spermatozoa. Acrosome reaction, binding to zona pellucida and fusion with the oolemma were lower in Prdx6 -/- capacitated spermatozoa than WT capacitated controls and lower in WT spermatozoa treated with the PRDX6 inhibitor. In conclusion, the inhibition of the PRDX6 Ca2+-iPLA2 activity promotes an oxidative stress affecting viability, motility, and the ability of mouse spermatozoa to fertilize oocytes. Thus, PRDX6 has a critical role in the protection of the mouse spermatozoon against oxidative stress to assure fertilizing competence.
Project description:Burkitt lymphoma is a fast-growing tumor derived from germinal center B cells. It is mainly treated with aggressive chemotherapy, therefore novel therapeutic approaches are needed due to treatment toxicity and developing resistance. Disturbance of red-ox homeostasis has recently emerged as an efficient antitumor strategy. Peroxiredoxins (PRDXs) are thioredoxin-family antioxidant enzymes that scavenge cellular peroxides and contribute to red-ox homeostasis. PRDXs are robustly expressed in various malignancies and critically involved in cell proliferation, differentiation and apoptosis. To elucidate potential role of PRDXs in lymphoma, we studied their expression level in B cell-derived primary lymphoma cells as well as in cell lines. We found that PRDX1 and PRDX2 are upregulated in tumor B cells as compared with normal counterparts. Concomitant knockdown of PRDX1 and PRDX2 significantly attenuated the growth rate of lymphoma cells. Furthermore, in human Burkitt lymphoma cell lines, we isolated dimeric 2-cysteine peroxiredoxins as targets for SK053, a novel thiol-specific small-molecule peptidomimetic with antitumor activity. We observed that treatment of lymphoma cells with SK053 triggers formation of covalent PRDX dimers, accumulation of intracellular reactive oxygen species, phosphorylation of ERK1/2 and AKT and leads to cell cycle arrest and apoptosis. Based on site-directed mutagenesis and modeling studies, we propose a mechanism of SK053-mediated PRDX crosslinking, involving double thioalkylation of active site cysteine residues. Altogether, our results suggest that peroxiredoxins are novel therapeutic targets in Burkitt lymphoma and provide the basis for new approaches to the treatment of this disease.
Project description:Peroxiredoxins (PRDXs) are members of a highly conserved peroxidase family and maintain intracellular reactive oxygen species (ROS) homeostasis. The family members are expressed in most organisms and involved in various biological processes, such as cellular protection against ROS, inflammation, carcinogenesis, atherosclerosis, heart diseases, and metabolism. In mammals, six PRDX members have been identified and are subdivided into three subfamilies: typical 2-Cys (PRDX1, PRDX2, PRDX3, and PRDX4), atypical 2-Cys (PRDX5), and 1-Cys (PRDX6) subfamilies. Knockout mouse models of PRDXs have been developed to investigate their in vivo roles. This review presents an overview of the knockout mouse models of PRDXs with emphases on the biological and physiological changes of these model mice.
Project description:Chlamydia trachomatis is the most prevalent sexually transmitted bacterial infection. However, whether Chlamydia trachomatis has a negative impact on sperm quality and male fertility is still controversial. Herein, we report the effects on sperm quality of the in vitro exposure of spermatozoa to Chlamydia trachomatis, and also the effects of male genital infection on male fertility using an animal model. Human and mouse sperm were obtained from healthy donors and cauda epididimys from C57BL/6 mice, respectively. Highly motile human or mouse spermatozoa were in vitro exposed to C. trachomatis (serovar E or LGV) or C. muridarum, respectively. Then, sperm quality parameters were analyzed. Moreover, male fertility of Chlamydia muridarum infected male C57BL/6 mice was assessed. Human or murine sperm in vitro exposed to increasing bacterial concentrations or soluble factors from C. trachomatis or C. muridarum, respectively, did not show differences in sperm motility and viability, apoptosis, mitochondrial membrane potential, DNA fragmentation, ROS production and lipid peroxidation levels, when compared with control sperm (p?>?0.05). Moreover, no differences in fertility parameters (potency, fecundity, fertility index, pre- and post-implantation loss) were observed between control and infected males. In conclusion, our results indicate that Chlamydia spp. neither directly exerts deleterious effects on spermatozoa nor impairs male fertility.