Effects of Heparan sulfate acetyl-CoA: Alpha-glucosaminide N-acetyltransferase (HGSNAT) inactivation on the structure and function of epithelial and immune cells of the testis and epididymis and sperm parameters in adult mice.

Heparan sulfate (HS), an abundant component of the apical cell surface and basement membrane, belongs to the glycosaminoglycan family of carbohydrates covalently linked to proteins called heparan sulfate proteoglycans. After endocytosis, HS is degraded in the lysosome by several enzymes, including heparan-alpha-glucosaminide N-acetyltransferase (HGSNAT), and in its absence causes Mucopolysaccharidosis III type C (Sanfilippo type C). Since endocytosis occurs in epithelial cells of the testis and epididymis, we examined the morphological effects of Hgsnat inactivation in these organs. In the testis, Hgsnat knockout (Hgsnat-Geo) mice revealed statistically significant decrease in tubule and epithelial profile area of seminiferous tubules. Electron microscopy (EM) analysis revealed cross-sectional tubule profiles with normal and moderately to severely altered appearances. Abnormalities in Sertoli cells and blood-testis barrier and the absence of germ cells in some tubules were noted along with altered morphology of sperm, sperm motility parameters and a reduction in fertilization rates in vitro. Along with quantitatively increased epithelial and tubular profile areas in the epididymis, EM demonstrated significant accumulations of electrolucent lysosomes in the caput-cauda regions that were reactive for cathepsin D and prosaposin antibodies. Lysosomes with similar storage materials were also found in basal, clear and myoid cells. In the mid/basal region of the epithelium of caput-cauda regions of KO mice, large vacuolated cells, unreactive for cytokeratin 5, a basal cell marker, were identified morphologically as epididymal mononuclear phagocytes (eMPs). The cytoplasm of the eMPs was occupied by a gigantic lysosome suggesting an active role of these cells in removing debris from the epithelium. Some eMPs were found in proximity to T-lymphocytes, a feature of dendritic cells. Taken together, our results reveal that upon Hgsnat inactivation, morphological alterations occur to the testis affecting sperm morphology and motility parameters and abnormal lysosomes in epididymal epithelial cells, indicative of a lysosomal storage disease.

Improving standard practices in studies using results from basic human semen examination.

The purpose of this article is to provide an explanation of the background behind a checklist that declares the laboratory methods used in a scientific study. It focuses primarily on implementing laboratory procedures to yield reliable results in basic semen examinations. While the World Health Organization (WHO) and international standards provide recommendations for basic semen examination, manuscripts submitted to Andrology frequently lack transparency regarding the specific techniques used. In addition, the terminology used for semen examination results often fails to provide a clear definition of the groups under study. Furthermore, the WHO's reference limits are often misinterpreted as strict boundaries between fertility and infertility. It is important to note that valid clinical andrological diagnoses and treatments cannot rely solely on semen examination results; they require proper laboratory procedures as a foundation for diagnosing and treating male patients. Therefore, scientific journals should promote the adoption of robust laboratory practices and an accurate definition of patient groups. A checklist can facilitate the design of high-quality studies and the creation of informative publications. Further, it can help journals assess submitted manuscripts and improve the overall quality of their publications.

Lysophosphatidic Acid Signalling Regulates Human Sperm Viability via the Phosphoinositide 3-Kinase/AKT Pathway.

Lysophosphatidic acid (LPA) signalling is essential for maintaining germ cell viability during mouse spermatogenesis; however, its role in human spermatozoa is unknown. We previously demonstrated that peroxiredoxin 6 (PRDX6) calcium-independent phospholipase A2 (iPLA2) releases lysophospholipids such as LPA or arachidonic acid (AA) and that inhibiting PRDX6 iPLA2 activity impairs sperm cell viability. The exogenous addition of LPA bypassed the inhibition of PRDX6 iPLA2 activity and maintained the active phosphoinositide 3-kinase (PI3K)/AKT pathway. Here, we aimed to study PI3K/AKT pathway regulation via LPA signalling and protein kinases in maintaining sperm viability. The localization of LPARs in human spermatozoa was determined using immunocytochemistry, and P-PI3K and P-AKT substrate phosphorylations via immunoblotting. Sperm viability was determined using the hypo-osmotic swelling test. LPAR1, 3, 5 and 6 were located on the sperm plasma membrane. The inhibition of LPAR1-3 with Ki16425 promoted the impairment of sperm viability and decreased the phosphorylation of PI3K AKT substrates. Inhibitors of PKC, receptor-type PTK and PLC impaired sperm viability and the PI3K/AKT pathway. Adding 1-oleoyl-2-acetyl-snglycerol (OAG), a cell-permeable analog of diacylglycerol (DAG), prevented the loss of sperm viability and maintained the phosphorylation of PI3K. In conclusion, human sperm viability is supported by LPAR signalling and regulated by PLC, PKC and RT-PTK by maintaining phosphorylation levels of PI3K and AKT substrates.

Changes of the Protein CoAlation Pattern in Response to Oxidative Stress and Capacitation in Human Spermatozoa.

The spermatozoa have limited antioxidant defences, a high polyunsaturated fatty acids content and the impossibility of synthesizing proteins, thus being susceptible to oxidative stress. High levels of reactive oxygen species (ROS) harm human spermatozoa, promoting oxidative damage to sperm lipids, proteins and DNA, leading to infertility. Coenzyme A (CoA) is a key metabolic integrator in all living cells. Recently, CoA was shown to function as a major cellular antioxidant mediated by a covalent modification of surface-exposed cysteines by CoA (protein CoAlation) under oxidative or metabolic stresses. Here, the profile of protein CoAlation was examined in sperm capacitation and in human spermatozoa treated with different oxidizing agents (hydrogen peroxide, (H2O2), diamide and tert-butyl hydroperoxide (t-BHP). Sperm viability and motility were also investigated. We found that H2O2 and diamide produced the highest levels of protein CoAlation and the greatest reduction of sperm motility without impairing viability. Protein CoAlation levels are regulated by 2-Cys peroxiredoxins (PRDXs). Capacitated spermatozoa showed lower levels of protein CoAlation than non-capacitation cells. This study is the first to demonstrate that PRDXs regulate protein CoAlation, which is part of the antioxidant response of human spermatozoa and participates in the redox regulation associated with sperm capacitation.

Sperm DNA methylome abnormalities occur both pre- and post-treatment in men with Hodgkin disease and testicular cancer.

BACKGROUND: Combination chemotherapy has contributed to increased survival from Hodgkin disease (HD) and testicular cancer (TC). However, questions concerning the quality of spermatozoa after treatment have arisen. While studies have shown evidence of DNA damage and aneuploidy in spermatozoa years following anticancer treatment, the sperm epigenome has received little attention. Our objectives here were to determine the impact of HD and TC, as well as their treatments, on sperm DNA methylation. Semen samples were collected from community controls (CC) and from men undergoing treatment for HD or TC, both before initiation of chemotherapy and at multiple times post-treatment. Sperm DNA methylation was assessed using genome-wide and locus-specific approaches. RESULTS: Imprinted gene methylation was not affected in the sperm of HD or TC men, before or after treatment. Prior to treatment, using Illumina HumanMethylation450 BeadChip (450 K) arrays, a subset of 500 probes was able to distinguish sperm samples from TC, HD and CC subjects; differences between groups persisted post-treatment. Comparing altered sperm methylation between HD or TC patients versus CC men, twice as many sites were affected in TC versus HD men; for both groups, the most affected CpGs were hypomethylated. For TC patients, the promoter region of GDF2 contained the largest region of differential methylation. To assess alterations in DNA methylation over time/post-chemotherapy, serial samples from individual patients were compared. With restriction landmark genome scanning and 450 K array analyses, some patients who underwent chemotherapy showed increased alterations in DNA methylation, up to 2 to 3 years post-treatment, when compared to the CC cohort. Similarly, a higher-resolution human sperm-specific assay that includes assessment of environmentally sensitive regions, or "dynamic sites," also demonstrated persistently altered sperm DNA methylation in cancer patients post-treatment and suggested preferential susceptibility of "dynamic" CpG sites. CONCLUSIONS: Distinct sperm DNA methylation signatures were present pre-treatment in men with HD and TC and may help explain increases in birth defects reported in recent clinical studies. Epigenetic defects in spermatozoa of some cancer survivors were evident even up to 2 years post-treatment. Abnormalities in the sperm epigenome both pre- and post-chemotherapy may contribute to detrimental effects on future reproductive health.

The Stallion Spermatozoa: A Valuable Model to Help Understand the Interplay Between Metabolism and Redox (De)regulation in Sperm Cells.

Significance: Proper functionality of the spermatozoa depends on the tight regulation of their redox status; at the same time these cells are highly energy demanding and in the energetic metabolism, principally in the electron transport chain in the mitochondria, reactive oxygen species are continuously produced, in addition to that observed in the Krebs cycle and during the ß-oxidation of fatty acids. Recent Advances: In addition, in glycolysis, elimination of phosphate groups from glyceraldehyde 3-phosphate and dihydroxyacetone phosphate results in the byproducts glyoxal (G) and methylglyoxal (MG); these products are 2-oxoaldehydes. The presence of adjacent carbonyl groups makes them strong electrophiles that react with nucleophiles in proteins, lipids, and DNA, forming advanced glycation end products. Critical Issues: This mechanism is behind subfertility in diabetic patients; in the animal breeding industry, commercial extenders for stallion semen contain a supraphysiological concentration of glucose that promotes MG production, constituting a potential model of interest. Future Directions: Increasing our knowledge of sperm metabolism and its interactions with redox regulation may improve current sperm technologies in use, and shall provide new clues to understanding infertility in males. Moreover, stallion spermatozoa due to its accessibility, intense metabolism, and suitability for proteomics/metabolomic studies may constitute a suitable model for studying regulation of metabolism and interactions between metabolism and redox homeostasis. Antioxid. Redox Signal. 37, 521-537.

Peroxiredoxin 6 Peroxidase and Ca2+-Independent Phospholipase A2 Activities Are Essential to Support Male-Mouse Fertility.

Human infertility is an important health problem that affects one in six couples worldwide. Half of these cases are due to male infertility. Oxidative stress is a common culprit of male infertility, promoting lipid peroxidation and the oxidation of proteins and DNA in spermatozoa, thereby impairing motility, capacitation and fertilization. Peroxiredoxin 6 (PRDX6) possesses peroxidase and Ca2+-independent-phospholipase-A2 (iPLA2) activities that scavenge ROS and repair oxidized sperm membranes, respectively. PRDX6 protects spermatozoa against oxidative stress. Infertile men's spermatozoa have impaired motility, elevated lipid peroxidation levels and DNA damage due to low PRDX6 levels. A lack of PRDX6 is associated with male-mouse infertility. Here, we determined the impact of the absence of PRDX6 peroxidase or iPLA2 activities on male-mouse fertility. Two-month-old male C57Bl6/J (wild-type), Prdx6-/-, C47S and D140A knock-in (peroxidase- and iPLA2-deficient, respectively) male mice were challenged with an in vivo oxidative stress triggered by tert-butyl hydroperoxide (t-BHP). C47S and D140A males produced smaller litters compared to wild-type controls. The t-BHP treatment promoted a lower number of pups, high levels of lipid peroxidation, tyrosine nitration, and DNA oxidation in all mutant spermatozoa compared to wild-type controls. All mutant spermatozoa had impaired capacitation and motility. In summary, both PRDX6 peroxidase and iPLA2 activities are essential to support male-mouse fertility.

Redox Regulation to Modulate Phosphorylation Events in Human Spermatozoa.

Significance: Spermatozoa are complex and compartmentalized cells that undergo capacitation, a series of biochemical and morphological changes to acquire the ability to fertilize oocytes. Reactive oxygen species (ROS) have a prominent dual role in capacitation. At physiological levels, ROS regulate numerous cellular processes, including increases of cyclic adenosine monophosphate, calcium, and activation of phosphorylation events needed for capacitation. On the contrary, at high concentrations that do not impair sperm viability, ROS can cause loss of motility and inhibition of capacitation. Higher ROS concentrations promote oxidation of lipids, proteins, and DNA leading to cell death, and these damages have been associated with male infertility. Critical Issues: When incubated under specific conditions, spermatozoa can produce low and controlled amounts of ROS that are not harmful but instead regulate numerous cellular processes, including the phosphorylation of tyrosine, serine, and threonine residues in critical proteins needed for sperm capacitation. Here, we outline the complex redox signaling in human spermatozoa needed to achieve fertility and the role of ROS as physiological mediators that trigger phosphorylation cascades. Moreover, we illustrate the importance of various phosphoproteins in spermatozoa capacitation, viability, and hyperactive motility. Future Directions: Further studies to elucidate the different phosphorylation players during sperm capacitation and acrosome reaction (the regulated exocytotic event that releases proteolytic enzymes allowing the spermatozoon to penetrate the zona pellucida and fertilize the oocyte) are essential to understand how the spermatozoon acquires the fertilizing ability to fertilize the oocyte. This knowledge will serve to develop novel diagnostic tools and therapy for male infertility. Antioxid. Redox Signal. 37, 437-450.

A Novel Combination of γ-Tocopherol-Rich Mixture of Tocopherols and Ascorbic Acid Restores Fertility in Cases of Tyrosine Nitration-Associated Male Infertility in Mice.

Infertility is an important health problem that affects up to 16% of couples worldwide. Male infertility is responsible for 50% of the cases. Currently, a physical examination, hormone profiling and the evaluation of two consecutive semen samples (to determine the sperm concentration, motility, morphology and, in very few cases, sperm DNA integrity) are the sole tools that physicians have to evaluate infertility in men. Antioxidant therapy is often used to improve sperm quality and function in infertile men. However, there are controversial results regarding the efficacy of these treatments. Prdx6-/- male mice are subfertile, displaying significant oxidative damage in the lipids, proteins and DNA of their spermatozoa. Here, we used Prdx6-/- male mice to test whether a novel combination of tocopherols that contained 60% γ-tocopherol and ascorbic acid could restore their fertility. These mice were fed with the supplemented (Vit. Mix) or control diets. To assess sperm quality, we determined the motility, levels of lipid peroxidation, DNA oxidation and tyrosine nitration in the spermatozoa. The number of pups sired by the Prdx6-/- mice fed with the Vit. Mix diet was higher than that sired by the males fed with the control diet, and the pups' mortality was lower. The sperm quality was improved in the males fed with the supplemented diet. We concluded that treatment with a supplement composed of tocopherols and rich in γ-tocopherol and ascorbic acid is effective in restoring fertility in cases where oxidative stress and high levels of tyrosine nitration are associated with male infertility.

Reactive Oxygen Species and Male Fertility.

Human infertility affects ~15% of couples worldwide, and it is now recognized that in half of these cases, the causes of infertility can be traced to men[...].

Long-Term Adverse Effects of Oxidative Stress on Rat Epididymis and Spermatozoa.

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.

Antioxidant Enzymes and Male Fertility: Lessons from Knockout Models.

Significance: Spermatozoa are very sensitive to high levels of reactive oxygen species (ROS) due to the limited antioxidant systems present in these terminal cells. However, tight regulation of ROS levels must be ensured to accomplish the unique goal of the spermatozoon, that is, the transfer of the paternal genome into the mature oocyte during the fertilization process. Thus, it is essential that the restricted antioxidant enzymatic systems are active for sperm function. Recent Advances: Oxidative stress is associated with low sperm quality. High levels of ROS in spermatozoa produce oxidation of lipids, proteins, and DNA that lead to lipid peroxidation, oxidation of essential structural proteins and enzymes, and mutations due to oxidation of DNA. Critical Issues: In this study, we described the available knockout mouse models that helped to better understand the role of different antioxidant enzymes in male fertility. We focused mainly on those studies that directly explore the effects of the lack of these enzymes in male fertility and included information when existing knockout mouse models produced for other purposes were used. Special attention was given in this review to the consequences of the absence of antioxidant enzymes on sperm quality and fertility of aging males from the knockout models. Future Directions: Further studies using novel mouse models lacking different antioxidants and their combinations are essential to understand the consequences of high levels of ROS in aging testes, epididymes, spermatozoa, and embryo development to produce a healthy baby.

GSTO2 Isoforms Participate in the Oxidative Regulation of the Plasmalemma in Eutherian Spermatozoa during Capacitation.

In addition to perinuclear theca anchored glutathione-s-transferase omega 2 (GSTO2), whose function is to participate in sperm nuclear decondensation during fertilization (Biol Reprod. 2019, 101:368-376), we herein provide evidence that GSTO2 is acquired on the sperm plasmalemma during epididymal maturation. This novel membrane localization was reinforced by the isolation and identification of biotin-conjugated surface proteins from ejaculated and capacitated boar and mouse spermatozoa, prompting us to hypothesize that GSTO2 has an oxidative/reductive role in regulating sperm function during capacitation. Utilizing an inhibitor specific to the active site of GSTO2 in spermatozoa, inhibition of this enzyme led to a decrease in tyrosine phosphorylation late in the capacitation process, followed by an expected decrease in acrosome exocytosis and motility. These changes were accompanied by an increase in reactive oxygen species (ROS) levels and membrane lipid peroxidation and culminated in a significant decrease in the percentage of oocytes successfully penetrated by sperm during in vitro fertilization. We conclude that GSTO2 participates in the regulation of sperm function during capacitation, most likely through protection against oxidative stress on the sperm surface.

Redox Regulation and Oxidative Stress: The Particular Case of the Stallion Spermatozoa.

Redox regulation and oxidative stress have become areas of major interest in spermatology. Alteration of redox homeostasis is recognized as a significant cause of male factor infertility and is behind the damage that spermatozoa experience after freezing and thawing or conservation in a liquid state. While for a long time, oxidative stress was just considered an overproduction of reactive oxygen species, nowadays it is considered as a consequence of redox deregulation. Many essential aspects of spermatozoa functionality are redox regulated, with reversible oxidation of thiols in cysteine residues of key proteins acting as an "on-off" switch controlling sperm function. However, if deregulation occurs, these residues may experience irreversible oxidation and oxidative stress, leading to malfunction and ultimately death of the spermatozoa. Stallion spermatozoa are "professional producers" of reactive oxygen species due to their intense mitochondrial activity, and thus sophisticated systems to control redox homeostasis are also characteristic of the spermatozoa in the horse. As a result, and combined with the fact that embryos can easily be collected in this species, horses are a good model for the study of redox biology in the spermatozoa and its impact on the embryo.

Peroxiredoxin 6 regulates the phosphoinositide 3-kinase/AKT pathway to maintain human sperm viability.

Peroxiredoxins (PRDXs) are antioxidant enzymes proven to control the levels of reactive oxygen species (ROS) and to avoid oxidative damage in the spermatozoon. Previously, we have shown that low amounts of PRDXs are associated with male infertility and that PRDX6 is the primary antioxidant defense in human spermatozoa, maintaining survival and DNA integrity (Gong et al., 2012, Fernandez and O'Flaherty, 2018). Oxidative stress can trigger different pathway cascades in the spermatozoa, including truncated apoptosis. It has been reported that the phosphorylation status of phosphoinositide 3-kinase (PI3K) and its target AKT (protein kinase B) prevent the spermatozoon from entering the truncated apoptotic cascade. Here, we aim to study the regulation of the PI3K/AKT pathway by PRDX6 and assess its role in maintaining sperm viability. Human semen samples were obtained over 1 year from 20 healthy non-smoking volunteers aged 22-30 years. Sperm viability, lipid peroxidation and apoptosis-like changes were determined by flow cytometry while phosphorylation of PI3K and AKT substrates were assessed by immunoblotting using anti-phospho-PI3K and anti-phospho-AKT substrates antibodies. We found that the addition of arachidonic acid and lysophosphatidic acid, products of PRDX6 calcium-independent phospholipase A2 (Ca2+-iPLA2), prevented loss of sperm viability and maintained the phosphorylation of PI3K. Antioxidant compounds such as D-penicillamine partially prevented the oxidative damage on spermatozoa that led to a reduction of their viability. Thus, other pathways can also participate in sperm survival and be regulated by PRDXs. In conclusion, PRDX6 contributes to the regulation of ROS production and the PI3K/AKT pathway for the maintenance of sperm survival.

Snap29 mutant mice recapitulate neurological and ophthalmological abnormalities associated with 22q11 and CEDNIK syndrome.

Synaptosomal-associated protein 29 (SNAP29) encodes a member of the SNARE family of proteins implicated in numerous intracellular protein trafficking pathways. SNAP29 maps to the 22q11.2 region and is deleted in 90% of patients with 22q11.2 deletion syndrome (22q11.2DS). Moreover, bi-allelic SNAP29 mutations in patients are responsible for CEDNIK (cerebral dysgenesis, neuropathy, ichthyosis, and keratoderma) syndrome. A mouse model that recapitulates abnormalities found in these syndromes is essential for uncovering the cellular basis of these disorders. In this study, we report that mice with a loss of function mutation of Snap29 on a mixed CD1;FvB genetic background recapitulate skin abnormalities associated with CEDNIK, and also phenocopy neurological and ophthalmological abnormalities found in CEDNIK and a subset of 22q11.2DS patients. Our work also reveals an unanticipated requirement for Snap29 in male fertility and supports contribution of hemizygosity for SNAP29 to the phenotypic spectrum of abnormalities found in 22q11.2DS patients.

Protective Role of Peroxiredoxins against Reactive Oxygen Species in Neonatal Rat Testicular Gonocytes.

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.

Peroxiredoxin 6: The Protector of Male Fertility.

The spermatozoon is a terminal cell with the unique purpose of delivering the paternal genome to the oocyte during fertilization. Once spermatozoa enter into the female reproductive tract, they count on only the antioxidant protection that they received during spermatogenesis and epididymal maturation. Peroxiredoxins (PRDXs), particularly PRDX6, are important players in the antioxidant protection and regulation of reactive oxygen species (ROS) levels in spermatozoa. PRDX6, through its peroxidase and calcium-independent phospholipase A2 activities, plays a major role in the regulation of ROS to maintain viability and motility and allow the spermatozoon to achieve fertilizing ability during the complex process of capacitation. The absence of PRDX6 is sufficient to promote abnormal reproductive outcomes in mice that resemble what we observe in infertile men. Indeed, Prdx6-/- spermatozoa display low motility and severe DNA damage, which is translated into reduced ability to fertilize oocytes in vitro or produce a low number of pups compared to wild-type controls. This review focuses on the role of PRDX6 as the primary antioxidant enzyme that protects the spermatozoon from oxidative-stress-associated damages to protect the paternal genome and assure fertility.

Peroxiredoxin 6 is the primary antioxidant enzyme for the maintenance of viability and DNA integrity in human spermatozoa.

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.

Ubiquitin Ligase Huwe1 Modulates Spermatogenesis by Regulating Spermatogonial Differentiation and Entry into Meiosis.

Spermatogenesis consists of a series of highly regulated processes that include mitotic proliferation, meiosis and cellular remodeling. Although alterations in gene expression are well known to modulate spermatogenesis, posttranscriptional mechanisms are less well defined. The ubiquitin proteasome system plays a significant role in protein turnover and may be involved in these posttranscriptional mechanisms. We previously identified ubiquitin ligase Huwe1 in the testis and showed that it can ubiquitinate histones. Since modulation of histones is important at many steps in spermatogenesis, we performed a complete characterization of the functions of Huwe1 in this process by examining the effects of its inactivation in the differentiating spermatogonia, spermatocytes and spermatids. Inactivation of Huwe1 in differentiating spermatogonia led to their depletion and formation of fewer pre-leptotene spermatocytes. The cell degeneration was associated with an accumulation of DNA damage response protein γH2AX, impaired downstream signalling and apoptosis. Inactivation of Huwe1 in spermatocytes indicated that Huwe1 is not essential for meiosis and spermiogenesis, but can result in accumulation of γH2AX. Collectively, these results provide a comprehensive survey of the functions of Huwe1 in spermatogenesis and reveal Huwe1's critical role as a modulator of the DNA damage response pathway in the earliest steps of spermatogonial differentiation.