The longitudinal loss of islet autoantibody responses from diagnosis of type 1 diabetes occurs progressively over follow-up and is determined by low autoantibody titres, early-onset, and genetic variants.

The clinical usefulness of post-diagnosis islet autoantibody levels is unclear and factors that drive autoantibody persistence are poorly defined in type 1 diabetes (T1D). Our aim was to characterise the longitudinal loss of islet autoantibody responses after diagnosis in a large, prospectively sampled UK cohort. Participants with T1D [n = 577] providing a diagnosis sample [range -1.0 to 2.0 years] and at least one post-diagnosis sample (<32.0 years) were tested for autoantibodies to glutamate decarboxylase 65 (GADA), islet antigen-2 (IA-2A), and zinc transporter 8 (ZnT8A). Select HLA and non-HLA SNPs were considered. Non-genetic and genetic factors were assessed by multivariable logistic regression models for autoantibody positivity at initial sampling and autoantibody loss at final sampling. For GADA, IA-2A, and ZnT8A, 70.8%, 76.8%, and 40.1%, respectively, remained positive at the final sampling. Non-genetic predictors of autoantibody loss were low baseline autoantibody titres (P < 0.0001), longer diabetes duration (P < 0.0001), and age-at-onset under 8 years (P < 0.01--0.05). Adjusting for non-genetic covariates, GADA loss was associated with low-risk HLA class II genotypes (P = 0.005), and SNPs associated with autoimmunity RELA/11q13 (P = 0.017), LPP/3q28 (P = 0.004), and negatively with IFIH1/2q24 (P = 0.018). IA-2A loss was not associated with genetic factors independent of other covariates, while ZnT8A loss was associated with the presence of HLA A*24 (P = 0.019) and weakly negatively with RELA/11q13 (P = 0.049). The largest longitudinal study of islet autoantibody responses from diagnosis of T1D shows that autoantibody loss is heterogeneous and influenced by low titres at onset, longer duration, earlier age-at-onset, and genetic variants. These data may inform clinical trials where post-diagnosis participants are recruited.

Paternal impacts on development: identification of genomic regions vulnerable to oxidative DNA damage in human spermatozoa.

STUDY QUESTION: Do all regions of the paternal genome within the gamete display equivalent vulnerability to oxidative DNA damage? SUMMARY ANSWER: Oxidative DNA damage is not randomly distributed in mature human spermatozoa but is instead targeted, with particular chromosomes being especially vulnerable to oxidative stress. WHAT IS KNOWN ALREADY: Oxidative DNA damage is frequently encountered in the spermatozoa of male infertility patients. Such lesions can influence the incidence of de novo mutations in children, yet it remains to be established whether all regions of the sperm genome display equivalent susceptibility to attack by reactive oxygen species. STUDY DESIGN, SIZE, DURATION: Human spermatozoa obtained from normozoospermic males (n = 8) were split into equivalent samples and subjected to either hydrogen peroxide (H2O2) treatment or vehicle controls before extraction of oxidized DNA using a modified DNA immunoprecipitation (MoDIP) protocol. Specific regions of the genome susceptible to oxidative damage were identified by next-generation sequencing and validated in the spermatozoa of normozoospermic males (n = 18) and in patients undergoing infertility evaluation (n = 14). PARTICIPANTS/MATERIALS, SETTING, METHODS: Human spermatozoa were obtained from normozoospermic males and divided into two identical samples prior to being incubated with either H2O2 (5 mm, 1 h) to elicit oxidative stress or an equal volume of vehicle (untreated controls). Alternatively, spermatozoa were obtained from fertility patients assessed as having high basal levels of oxidative stress within their spermatozoa. All semen samples were subjected to MoDIP to selectively isolate oxidized DNA, prior to sequencing of the resultant DNA fragments using a next-generation whole-genomic sequencing platform. Bioinformatic analysis was then employed to identify genomic regions vulnerable to oxidative damage, several of which were selected for real-time quantitative PCR (qPCR) validation. MAIN RESULTS AND THE ROLE OF CHANCE: Approximately 9000 genomic regions, 150-1000 bp in size, were identified as highly vulnerable to oxidative damage in human spermatozoa. Specific chromosomes showed differential susceptibility to damage, with chromosome 15 being particularly sensitive to oxidative attack while the sex chromosomes were protected. Susceptible regions generally lay outside protamine- and histone-packaged domains. Furthermore, we confirmed that these susceptible genomic sites experienced a dramatic (2-15-fold) increase in their burden of oxidative DNA damage in patients undergoing infertility evaluation compared to normal healthy donors. LIMITATIONS, REASONS FOR CAUTION: The limited number of samples analysed in this study warrants external validation, as do the implications of our findings. Selection of male fertility patients was based on high basal levels of oxidative stress within their spermatozoa as opposed to specific sub-classes of male factor infertility. WIDER IMPLICATIONS OF THE FINDINGS: The identification of genomic regions susceptible to oxidation in the male germ line will be of value in focusing future analyses into the mutational load carried by children in response to paternal factors such as age, the treatment of male infertility using ART and paternal exposure to environmental toxicants. STUDY FUNDING/COMPETING INTEREST(S): Project support was provided by the University of Newcastle's (UoN) Priority Research Centre for Reproductive Science. M.J.X. was a recipient of a UoN International Postgraduate Research Scholarship. B.N. is the recipient of a National Health and Medical Research Council of Australia Senior Research Fellowship. Authors declare no conflict of interest.

Oxidative Damage to Sperm DNA: Attack and Defense.

Due to its particular "silent" metabolic state, without transcription or translation, and a low level of cytosolic protective activities, mature sperm is a cellular type of aerobic organisms particularly at risk of oxidative damage. Despite the efforts of the male genital tract to treat this problem, a subcellular compartment of the sperm, the nucleus, and consequently, the paternal DNA cannot be effectively protected. There is an accumulation of evidence that oxidative damage to sperm DNA is quite common in male infertilities/subfertilities with potential harmful impacts on reproductive success, including the transgenerational inheritance of a paternal chromosomal lot carrying mutations.

The serine protease testisin is present on the surface of capacitated stallion spermatozoa and interacts with key zona pellucida binding proteins.

BACKGROUND AND OBJECTIVES: Serine proteases are emerging as important players in the spermatozoon's acquisition of functional competence. This study aimed to characterize the serine protease testisin (PRSS21) in stallion spermatozoa, examining its surface expression, possible origins in the testis and epididymis, and changes in response to capacitation and acrosome reaction, as well as its capacity to form high molecular weight complexes and interact with other proteins. MATERIALS AND METHODS: The role of serine proteases in spontaneous capacitation and acrosome reaction of stallion spermatozoa was established using the serine protease inhibitor, AEBSF. Testisin localization, before and after exposure of stallion spermatozoa to capacitating conditions and calcium ionophore, was examined using live cell immunofluorescence and flow cytometry. Immunohistochemistry of testicular and epididymal tissues was used to further dissect the origins of sperm testisin. Testisin's participation in high molecular weight protein complexes and identification of its interacting partner proteins were investigated using Blue Native PAGE, co-immunoprecipitation, and mass spectrometry, with interrogation of protein-protein interaction databases and gene ontology analysis of partner proteins used to further explore the potential roles of the testisin-containing complex in sperm function. RESULTS: Testisin surface expression increased significantly in capacitated spermatozoa (p < 0.001), increased further following acrosome reaction (p < 0.01), and was localized to the equatorial region of the sperm head. Testisin was also detected in luminal fluid within the caput and corpus regions of the epididymis, epididymal spermatozoa, and epididymal epithelial cells. Testisin formed several multiprotein complexes; co-immunoprecipitation revealed interactions of testisin with a multitude of zona pellucida-binding proteins, including ZPBP, ZAN, acrosin, several heat-shock proteins, and components of the TCP1 complex. CONCLUSION: Testisin appears to form part of the zona pellucida-binding complex in stallion spermatozoa and may be involved in the proteolytic cascade that prepares the sperm surface for interaction with the oocyte.

Parabens generate reactive oxygen species in human spermatozoa.

Parabens are used as antimicrobial preservative agent in many commercial products including cosmetics and pharmaceuticals. Weak oestrogenic and antiandrogenic activities have been attributed to parabens in in vitro and in vivo studies. In this study, human spermatozoa were exposed to different concentrations of an equimolar paraben mixture containing methyl, ethyl, propyl and butylparaben as well as to methylparaben alone at a concentration that is typical of commercially available vaginal lubricants. The induction of oxidative stress and DNA damage was then assessed at different time points. Our results demonstrate that the paraben mixture was capable of stimulating the generation of mitochondrial and cytosolic reactive oxygen species (ROS), inhibiting sperm motility and viability in a dose-dependent manner. The ability of individual parabens to activate ROS generation and induce oxidative DNA damage was related to alkyl chain length. At the concentration used clinically, methylparaben inhibited sperm motility after both 2 and 5 h exposure (p < 0.05) and affected cell viability (p < 0.01) while augmenting ROS production and oxidative DNA damage. However, DNA fragmentation was not evident following methylparaben exposure. Based on these results, we conclude that, at the concentrations used in commercially available formulations, parabens may impair sperm motility, enhance the generation of mitochondrial ROS and stimulate the formation of oxidative DNA adducts. Taken together, these data underline the potential cytotoxic and genotoxic impact of such compounds in a clinical setting.

Accuracy of human sperm DNA oxidation quantification and threshold determination using an 8-OHdG immuno-detection assay.

STUDY QUESTION: Can a discriminant threshold be determined for human sperm DNA oxidation? SUMMARY ANSWER: A discriminant threshold was found with 65.8% of 8-hydroxy-2'-deoxyguanosine (8-OHdG)-positive sperm cells and a mean intensity of fluorescence (MIF) of 552 arbitrary units. WHAT IS KNOWN ALREADY: Oxidative stress is known to interfere with sperm quality and fertilizing capacity. However, current practice does not include the routine determination of oxidative DNA damage in spermatozoa; optimized consensus protocols are lacking and no thresholds of normality have been established. STUDY DESIGN, SIZE, DURATION: Intra- and inter-method comparisons between four protocols (I-IV) were conducted to determine the most relevant and efficient means of assessing human sperm 8-OHdG content. Tests of assay repeatability, specificity, sensitivity and stability were performed to validate an optimized methodology for routine diagnostic use. PARTICIPANTS/MATERIALS, SETTING, METHODS: This prospective study compared three immuno-detection methods including immunocytochemistry, fluorescence microscopy and flow cytometry. Sperm DNA oxidation for 80 patients was determined relative to semen parameters and clinical conditions, using the selected immuno-detection protocol in comparison with a commercial kit. These patients (age 35 ± 1 years: mean ± SEM) presented with normozoospermic (n = 40) or altered parameters (necro- or/and astheno- or/and teratozoospermia or/and leukocytospermia). MAIN RESULTS AND THE ROLE OF CHANCE: Significant positive Pearson and Spearman correlations were determined for 8-OHdG values and sperm parameters using protocol III. A notable high and positive correlation was revealed for MIF with BMI and leukocyte concentration. Protocol III was the most discriminating method regarding assay repeatability, specificity, sensitivity, stability and reliability for sperm parameter alterations, in particular leukocytospermia according to parametric or non-parametric tests, effect-size determinations and factorial analysis such as principal component analysis and factor discriminant analysis. Of interest is that 39% of the subjects with 'pathological' sperm DNA oxidation values were normozoospermic. LIMITATIONS, REASONS FOR CAUTION: The oligozoospermic population was not evaluated in this study because insufficient material was available to carry out the comparisons. However, spermatozoa concentration was taken into account in the statistical analysis. WIDER IMPLICATIONS OF THE FINDINGS: Our study is the first validation of a protocol to determine a discriminant threshold for human sperm DNA oxidation. The protocol's detection accuracy for 8-OHdG human sperm DNA residues, stability over time, and relationship to human sperm quality were demonstrated. The assay should find application in the diagnosis of male factor infertility associated with oxidative stress. STUDY FUNDING/COMPETING INTEREST(S): This work was funded by institutional grants from the CNRS, INSERM and Université Clermont Auvergne (to J.R.D.) and by Clermont-Ferrand Hospital-CECOS research funds (to L.J. and F.B.). P.G., A.M., R.J.A. and J.D. are, respectively, CEO, scientific director and scientific advisors of a US-based biotech company (Celloxess, Princeton, NJ, USA) involved in preventative medicine with a focus on the generation of antioxidant oral supplements.

A quarter of patients with type 1 diabetes have co-existing non-islet autoimmunity: the findings of a UK population-based family study.

Individuals with type 1 diabetes (T1D) are at increased risk of coeliac disease (CD), autoimmune thyroiditis and autoimmune gastritis, but the absolute risks are unclear. The aim of this study was to investigate the prevalence of autoantibodies to tissue transglutaminase (TGA), thyroid peroxidase (TPOA) and gastric H+ /K+ -ATPase (ATPA) and their genetic associations in a well-characterized population-based cohort of individuals with T1D from the Bart's-Oxford family study for whom islet autoantibody prevalence data were already available. Autoantibodies in sera from 1072 patients (males/females 604/468; median age 11·8 years, median T1D duration 2·7 months) were measured by radioimmunoassays; HLA class II risk genotype was analysed in 973 (91%) using polymerase chain reaction with sequence specific primers (PCR-SSP). The prevalence of TGA (and/or history of CD), TPOA and ATPA in patients was 9·0, 9·6 and 8·2%, respectively; 3·1% had two or more autoantibodies. Females were at higher risk of multiple autoimmunity; TGA/CD were associated with younger age and TPOA with older age. ATPA were uncommon in patients under 5 years, and more common in older patients. Anti-glutamate decarboxylase autoantibodies were predictive of co-existing TPOA/ATPA. TGA/CD were associated with human leucocyte antigen (HLA) DR3-DQ2, with the DR3-DQ2/DR3-DQ2 genotype conferring the highest risk, followed by DR4-DQ8/DR4-DQ8. ATPA were associated with DR3-DQ2, DRB1*0404 (in males) and the DR3-DQ2/DR4-DQ8 genotype. TPOA were associated with the DR3-DQ2/DR3-DQ2 genotype. Almost one-quarter of patients diagnosed with T1D aged under 21 years have at least one other organ-specific autoantibody. HLA class II genetic profiling may be useful in identifying those at risk of multiple autoimmunity.

The effects of radiofrequency electromagnetic radiation on sperm function.

Mobile phone usage has become an integral part of our lives. However, the effects of the radiofrequency electromagnetic radiation (RF-EMR) emitted by these devices on biological systems and specifically the reproductive systems are currently under active debate. A fundamental hindrance to the current debate is that there is no clear mechanism of how such non-ionising radiation influences biological systems. Therefore, we explored the documented impacts of RF-EMR on the male reproductive system and considered any common observations that could provide insights on a potential mechanism. Among a total of 27 studies investigating the effects of RF-EMR on the male reproductive system, negative consequences of exposure were reported in 21. Within these 21 studies, 11 of the 15 that investigated sperm motility reported significant declines, 7 of 7 that measured the production of reactive oxygen species (ROS) documented elevated levels and 4 of 5 studies that probed for DNA damage highlighted increased damage due to RF-EMR exposure. Associated with this, RF-EMR treatment reduced the antioxidant levels in 6 of 6 studies that discussed this phenomenon, whereas consequences of RF-EMR were successfully ameliorated with the supplementation of antioxidants in all 3 studies that carried out these experiments. In light of this, we envisage a two-step mechanism whereby RF-EMR is able to induce mitochondrial dysfunction leading to elevated ROS production. A continued focus on research, which aims to shed light on the biological effects of RF-EMR will allow us to test and assess this proposed mechanism in a variety of cell types.

Analysis of the effects of polyphenols on human spermatozoa reveals unexpected impacts on mitochondrial membrane potential, oxidative stress and DNA integrity; implications for assisted reproductive technology.

The need to protect human spermatozoa from oxidative stress during assisted reproductive technology, has prompted a detailed analysis of the impacts of phenolic compounds on the functional integrity of these cells. Investigation of 16 individual compounds revealed a surprising variety of negative effects including: (i) a loss of mitochondrial membrane potential (Δψm) via mechanisms that were not related to opening of the permeability transition pore but associated with a reduction in thiol expression, (ii) a decline in intracellular reduced glutathione, (iii) the stimulation of pro-oxidant activity including the induction of ROS generation from mitochondrial and non-mitochondrial sources, (iv) stimulation of lipid peroxidation, (v) the generation of oxidative DNA damage, and (vi) impaired sperm motility. For most of the polyphenolic compounds examined, the loss of motility was gradual and highly correlated with the induction of lipid peroxidation (r=0.889). The exception was gossypol, which induced a rapid loss of motility due to its inherent alkylating activity; one consequence of which was a marked reduction in carboxymethyl lysine expression on the sperm tail; a post-translational modification that is known to play a key role in the regulation of sperm movement. The only polyphenols that did not appear to have adverse effects on spermatozoa were resveratrol, genistein and THP at doses below 100µM. These compounds could, therefore, have some therapeutic potential in a clinical setting.

D-penicillamine prevents ram sperm agglutination by reducing the disulphide bonds of a copper-binding sperm protein.

Head-to-head agglutination of ram spermatozoa is induced by dilution in the Tyrode's capacitation medium with albumin, lactate and pyruvate (TALP) and ameliorated by the addition of the thiol d-penicillamine (PEN). To better understand the association and disassociation of ram spermatozoa, we investigated the mechanism of action of PEN in perturbing sperm agglutination. PEN acts as a chelator of heavy metals, an antioxidant and a reducing agent. Chelation is not the main mechanism of action, as the broad-spectrum chelator ethylenediaminetetraacetic acid and the copper-specific chelator bathocuproinedisulfonic acid were inferior anti-agglutination agents compared with PEN. Oxidative stress is also an unlikely mechanism of sperm association, as PEN was significantly more effective in ameliorating agglutination than the antioxidants superoxide dismutase, ascorbic acid, α-tocopherol and catalase. Only the reducing agents cysteine and DL-dithiothreitol displayed similar levels of non-agglutinated spermatozoa at 0 h compared with PEN but were less effective after 3 h of incubation (37 °C). The addition of 10 µM Cu(2+) to 250 µM PEN + TALP caused a rapid reversion of the motile sperm population from a non-agglutinated state to an agglutinated state. Other heavy metals (cobalt, iron, manganese and zinc) did not provoke such a strong response. Together, these results indicate that PEN prevents sperm association by the reduction of disulphide bonds on a sperm membrane protein that binds copper. ADAM proteins are possible candidates, as targeted inhibition of the metalloproteinase domain significantly increased the percentage of motile, non-agglutinated spermatozoa (52.0% ± 7.8) compared with TALP alone (10.6% ± 6.1).

A novel antioxidant formulation designed to treat male infertility associated with oxidative stress: promising preclinical evidence from animal models.

STUDY QUESTION: Does a novel antioxidant formulation designed to restore redox balance within the male reproductive tract, reduce sperm DNA damage and increase pregnancy rates in mouse models of sperm oxidative stress? SUMMARY ANSWER: Oral administration of a novel antioxidant formulation significantly reduced sperm DNA damage in glutathione peroxidase 5 (GPX5), knockout mice and restored pregnancy rates to near-normal levels in mice subjected to scrotal heat stress. WHAT IS KNOWN ALREADY: Animal and human studies have documented the adverse effect of sperm DNA damage on fertilization rates, embryo quality, miscarriage rates and the transfer of de novo mutations to offspring. Semen samples of infertile men are known to be deficient in several key antioxidants relative to their fertile counterparts. Antioxidants alone or in combination have demonstrated limited efficacy against sperm oxidative stress and DNA damage in numerous human clinical trials, however these studies have not been definitive and an optimum combination has remained elusive. STUDY DESIGN, SIZE, DURATION: The efficacy of the antioxidant formulation was evaluated in two well-established mouse models of oxidative stress, scrotal heating and Gpx5 knockout (KO) mice, (n = 12 per experimental group), by two independent laboratories. Mice were provided the antioxidant product in their drinking water for 2-8 weeks and compared with control groups for sperm DNA damage and pregnancy rates. PARTICIPANTS/MATERIALS, SETTING, METHODS: In the Gpx5 KO model, oxidative DNA damage was monitored in spermatozoa by immunocytochemical detection of 8-hydroxy-2'-deoxyguanosine (8OHdG). In the scrotal heat stress model, male fertility was tested by partnering with three females for 5 days. The percentage of pregnant females, number of vaginal plugs, resorptions per litter, and litter size were recorded. MAIN RESULTS AND ROLE OF CHANCE: Using immunocytochemical detection of 8OHdG as a biomarker of DNA oxidation, analysis of control mice revealed that around 30% of the sperm population was positively stained. This level increased to about 60% in transgenic mice deficient in the antioxidant enzyme, GPX5. Our results indicate that an 8 week pretreatment of Gpx5 KO mice with the antioxidant formulation provided complete protection of sperm DNA against oxidative damage. In mouse models of scrotal heat stress, only 35% (19/54) of female mice became pregnant resulting in 169 fetuses with 18% fetal resorption (30/169). This is in contrast to the antioxidant pretreated group where 74% (42/57) of female mice became pregnant, resulting in 427 fetuses with 9% fetal resorption (38/427). In both animal models the protection provided by the novel antioxidant was statistically significant (P < 0.01 for the reduction of 8OHdG in the spermatozoa of Gpx5 KO mice and P < 0.05 for increase in fertility in the scrotal heat stress model). LIMITATIONS, REASONS FOR CAUTION: It was not possible to determine the exact level of antioxidant consumption for each mouse during the treatment period. WIDER IMPLICATIONS OF THE FINDINGS: Recent clinical studies confirm moderate to severe sperm DNA damage in about 60% of all men visiting IVF centers and in about 80% of men diagnosed with idiopathic male infertility. Our results, if confirmed in humans, will impact clinical fertility practice because they support the concept of using an efficacious antioxidant supplementation as a preconception therapy, in order to optimize fertilization rates, help to maintain a healthy pregnancy and limit the mutational load carried by children. STUDY FUNDING/COMPETING INTERESTS: The study was funded by the Clermont Université and the University of Madrid. P.G. is the Managing Director of CellOxess LLC, which has a commercial interest in the detection and resolution of oxidative stress. A.M. and A.P. are employees of CellOxess, LLC. J.R.D., A.G.-A. and R.J.A. are honorary members of the CellOxess advisory board.

Involvement of homocysteine, homocysteine thiolactone, and paraoxonase type 1 (PON-1) in the etiology of defective human sperm function.

This study reports, for the first time, the significant (p ≤ 0.01) accumulation of homocysteine residues in low density, defective sperm suspensions isolated from patients attending an infertility clinic. This overabundance of homocysteine was not related to a deficiency in folate availability but may have been a reflection of the oxidative stress that characterizes such defective sperm populations. Direct addition of the homocysteine cyclic congener, homocysteine thiolactone, to human spermatozoa resulted in the rapid induction of mitochondrial reactive oxygen species (ROS) generation (p < 0.001), the stimulation of lipid peroxidation (p < 0.01), the promotion of tyrosine phosphorylation (p < 0.001), and the suppression of sperm motility (p < 0.001) in the absence of any significant impact on DNA integrity. The parent homocysteine molecule was less active and took 24 h to stimulate mitochondrial ROS production possibly because of the need to convert this compound to the corresponding thiolactone before it could exert a measureable biological effect. Thiolactone was also effective in suppressing the carboxymethylation of key proteins in the sperm tail, which are thought to be involved in the regulation of sperm movement. The major enzyme responsible for removing thiolactone from proteins, paraoxonase (PON-1), was shown to be a major target for alkylation by lipid aldehydes, such as 4-hydroxynonenal, generated as a consequence of oxidative stress. Exposure of human spermatozoa to such aldehydes resulted in a dose-dependent accumulation of homocysteine in spermatozoa (p < 0.03). These results suggest that one of the consequences of oxidative stress in mammalian spermatozoa is the inhibition of PON-1, which then enhances the availability of homocysteine thiolactone to interact with the epsilon-amino group of lysine residues on sperm proteins, triggering a raft of significant biological changes in these cells that ultimately compromise sperm function.

Penicillamine prevents ram sperm agglutination in media that support capacitation.

Ram spermatozoa are difficult to capacitate in vitro. Here we describe a further complication, the unreported phenomenon of head-to-head agglutination of ram spermatozoa following dilution in the capacitation medium Tyrodes plus albumin, lactate and pyruvate (TALP). Sperm agglutination is immediate, specific and persistent and is not associated with a loss of motility. Agglutination impedes in vitro sperm handling and analysis. So the objectives of this study were to investigate the cause of sperm agglutination and potential agents which may reduce agglutination. The percentage of non-agglutinated, motile spermatozoa increased when bicarbonate was omitted from complete TALP suggesting that bicarbonate ions stimulate the agglutination process. d-penicillamine (PEN), a nucleophilic thiol, was highly effective at reducing agglutination. The inclusion of 250 µM PEN in TALP reduced the incidence of motile, agglutinated spermatozoa from 76.7 ± 2.7% to 2.8 ± 1.4%. It was then assessed if PEN (1 mM) could be included in existing ram sperm capacitation protocols (TALP +1 mM dibutyryl cAMP, caffeine and theophylline) to produce spermatozoa that were simultaneously capacitated and non-agglutinated. This protocol resulted in a sperm population which displayed high levels of tyrosine phosphorylated proteins and lipid disordered membranes (merocyanine-540) while remaining motile, viable, acrosome-intact and non-agglutinated. In summary, PEN (1 mM) can be included in ram sperm capacitation protocols to reduce sperm agglutination and allow for the in vitro assessment of ram sperm capacitation.

Human spermatozoa possess an IL4I1 l-amino acid oxidase with a potential role in sperm function.

Reactive oxygen species (ROS) are known to play an important role in the regulation of human sperm function. In this study, we demonstrate for the first time that human spermatozoa possess interleukin-induced gene 1 (IL4I1), an l-amino acid oxidase (LAAO) which is capable of generating ROS on exposure to aromatic amino acids in the presence of oxygen. The preferred substrates were found to be phenylalanine and tryptophan while the enzyme was located in the acrosomal region and midpiece of these cells. In contrast to equine and bovine spermatozoa, enzyme activity was lost as soon as the spermatozoa became non-viable. On a cell-to-cell basis human spermatozoa were also shown to generate lower levels of hydrogen peroxide than their equine counterparts on exposure to phenylalanine. Stimulation of LAAO activity resulted in the induction of several hallmarks of capacitation including tyrosine phosphorylation of the sperm flagellum and concomitant activation of phospho-SRC expression. In addition, stimulation of LAAO resulted in an increase in the levels of acrosomal exocytosis in both the presence and absence of progesterone stimulation, via mechanisms that could be significantly reversed by the presence of catalase. As is often the case with free radical-mediated phenomena, prolonged exposure of human spermatozoa to phenylalanine resulted in the stimulation of apoptosis as indicated by significant increases in mitochondrial superoxide generation and the activation of intracellular caspases. These results confirm the existence of an LAAO in human spermatozoa with a potential role in driving the redox regulation of sperm capacitation and acrosomal exocytosis.

Stallion fertility: a focus on the spermatozoon.

Stallion fertility is a vast subject, with a wide array of permutations that can impact reproductive performance in either positive or negative ways. This review is intended to address a mere segment of the male fertility issue, but the very essence of the male contribution to fertilisation, that of the spermatozoon. Spermatozoal ultrastructure and form-to-function are detailed and spermatozoal metabolism is discussed, with specific reference to distinctive characteristics of stallion spermatozoa. Lastly, methods for assessment of spermatozoal function are considered, with emphasis on spermatozoal motility, the acrosome reaction and spermatozoon-oocyte interactions. Closing comments address the need for development and standardisation of molecular-based assays for use with spermatozoa of stallions whose subfertility cannot be explained with conventional tests.

Potential importance of transition metals in the induction of DNA damage by sperm preparation media.

STUDY QUESTION: What are the mechanisms by which the preparation of spermatozoa on discontinuous density gradients leads to an increase in oxidative DNA damage? SUMMARY ANSWER: The colloidal silicon solutions that are commonly used to prepare human spermatozoa for assisted reproduction technology (ART) purposes contain metals in concentrations that promote free radical-mediated DNA damage. WHAT IS KNOWN ALREADY: Sporadic reports have already appeared indicating that the use of colloidal silicon-based discontinuous density gradients for sperm preparation is occasionally associated with the induction of oxidative DNA damage. The cause of this damage is however unknown. STUDY DESIGN, SIZE, DURATION: This study comprised a series of experiments designed to: (i) confirm the induction of oxidative DNA damage in spermatozoa prepared on commercially available colloidal silicon gradients, (ii) compare the levels of damage observed with alterative sperm preparation techniques including an electrophoretic approach and (iii) determine the cause of the oxidative DNA damage and develop strategies for its prevention. The semen samples employed for this analysis involved a cohort of >50 unselected donors and at least three independent samples were used for each component of the analysis. PARTICIPANTS/MATERIALS, SETTING, METHODS: The setting was a University biomedical science laboratory. The major techniques employed were: (i) flow cytometry to study reactive oxygen species generation, lipid peroxidation and DNA damage, (ii) computer-aided sperm analysis to measure sperm movement and (iii) inductively coupled mass spectrometry to determine the elemental composition of sperm preparation media. MAIN RESULTS AND THE ROLE OF CHANCE: Oxidative DNA damage is induced in spermatozoa prepared on PureSperm(®) discontinuous colloidal silicon gradients (P < 0.001 versus repeated centrifugation) because this medium contains metals, particularly Fe, Al and Cu, which are known to promote free radical generation in the immediate vicinity of DNA. This damage can be significantly accentuated by reducing agents, such as ascorbate (P < 0.001) and inhibited by selective chelation (P < 0.001). This problem is not confined to PureSperm(®); analysis of additional commercial sperm preparation media revealed that metal contamination is a relatively constant feature of such products. LIMITATIONS, REASONS FOR CAUTION: While the presence of metals, particularly transition metals, may exacerbate the levels of oxidative DNA damage seen in human spermatozoa, the significance of such damage has not yet been tested in suitably powered clinical trials. WIDER IMPLICATIONS OF THE FINDINGS: The results explain why the preparation of spermatozoa on discontinuous colloidal silicon gradients can result in oxidative DNA damage. The results are of immediate relevance to the development of safe, effective protocols for the preparation of spermatozoa for ART purposes. STUDY FUNDING/COMPETING INTERESTS: The study was funded by the Australian Health and Medical Research Council. One of the authors (R.J.A.) has had a consultantship with a biotechnology company, NuSep, interested in the development of electrophoretic methods of sperm preparation. He has no current financial interest in this area. None of the other authors have a conflict of interest to declare.

The senescence-accelerated mouse prone 8 as a model for oxidative stress and impaired DNA repair in the male germ line.

The discovery of a truncated base excision repair pathway in human spermatozoa mediated by OGG1 has raised questions regarding the effect of mutations in critical DNA repair genes on the integrity of the paternal genome. The senescence-accelerated mouse prone 8 (SAMP8) is a mouse model containing a suite of naturally occurring mutations resulting in an accelerated senescence phenotype largely mediated by oxidative stress, which is further enhanced by a mutation in the Ogg1 gene, greatly reducing the ability of the enzyme to excise 8-hydroxy,2'-deoxyguanosine (8OHdG) adducts. An analysis of the reproductive phenotype of the SAMP8 males revealed a high level of DNA damage in caudal epididymal spermatozoa as measured by the alkaline Comet assay. Furthermore, these lesions were confirmed to be oxidative in nature, as demonstrated by significant increases in 8OHdG adduct formation in the SAMP8 testicular tissue (P<0.05) as well as in mature spermatozoa (P<0.001) relative to a control strain (SAMR1). Despite this high level of oxidative DNA damage in spermatozoa, reactive oxygen species generation was not elevated and motility of spermatozoa was found to be similar to that for the control strain with the exception of progressive motility, which exhibited a slight but significant decline with advancing age (P<0.05). When challenged with Fenton reagents (H2O2 and Fe2+), the SAMP8 spermatozoa demonstrated a highly increased susceptibility to formation of 8OHdG adducts compared with the controls (P<0.001). These data highlight the role of oxidative stress and OGG1-dependent base excision repair mechanisms in defining the genetic integrity of mammalian spermatozoa.

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

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.