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.

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.

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 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.

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).

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.

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.

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.

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 source and significance of DNA damage in human spermatozoa; a commentary on diagnostic strategies and straw man fallacies.

This article considers the origins of DNA damage in human spermatozoa, the methods that are available to monitor this aspect of semen quality and the clinical significance of such measurements. DNA damage in spermatozoa appears to be largely oxidative in nature, inversely correlated with levels of nuclear protamination and frequently associated with the activation of a truncated apoptotic pathway. DNA base adducts formed as a result of oxidative attack are released from the spermatozoa into the extracellular space through the action of a glycosylase, OGG1. This creates an abasic site, which is not resolved until fertilization because spermatozoa do not possess the molecular machinery needed to continue the base excision repair pathway. The abasic sites so generated in human spermatozoa are readily detected by SCSA or the Comet assay; however, no signal is detectable with TUNEL. This is because spermatozoa lack the enzyme (APE1) needed to create the free 3' hydroxyl groups required by this detection system. Nevertheless, spermatozoa do eventually become TUNEL positive as they enter the perimortem. The American Society of Reproductive Medicine Practice Committee has suggested that DNA damage in spermatozoa should not be assessed because the correlation with pregnancy is inconsistent across independent studies. However, this is a straw man argument. The reason why such assays should be undertaken is not just that they reflect the underlying quality of spermatogenesis but, more importantly, that the DNA damage they reveal may have detrimental effects on the developmental normality of the embryo and the health of possible future children.

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.

Analysis of the global methylation status of human spermatozoa and its association with the tendency of these cells to enter apoptosis.

The methylation status of human spermatozoa has been examined in relation to the isopycnic density of these cells and their tendency to spontaneously default to an apoptotic state. DNA methylation was evaluated using three independent procedures: high-pressure liquid chromatography, flow cytometry and immunocytochemistry. All three techniques revealed that poor-quality spermatozoa recovered from the low-density region of Percoll gradients were characterised by a global hypermethylation of their DNA. Hypermethylation was visualised with an anti-5-methylcytosine antibody as punctate areas of cross-reactivity randomly distributed throughout the chromatin. Immunocytochemical evidence was also obtained suggesting that the sperm mitochondrial genome exists in a heavily methylated state, as a possible buffer against unscheduled transcription. Defective human spermatozoa were also shown to exhibit a tendency to default to an apoptotic state characterised by an increase in annexin V binding. The measurement of annexin V binding levels in individual sperm populations was found to be highly correlated with sperm vitality (P < 0.001) and the methylation status of their DNA (P < 0.001). We conclude that the generation of defective, apoptotic human spermatozoa is associated with disorders of spermatogenesis that lead to a global hypermethylation of their nuclear DNA.

Investigation of the expression and functional significance of the novel mouse sperm protein, a disintegrin and metalloprotease with thrombospondin type 1 motifs number 10 (ADAMTS10).

Fertilization represents the culmination of a series of complex interactions between male and female gametes. Despite advances in our understanding, the precise molecular mechanisms underlying these fundamental interactions remain largely uncharacterized. There is however growing recognition that this process requires the concerted action of multiple sperm receptors that possess affinity for complementary zona pellucida ligands and those that reside on the surface of the oolemma. Among the candidate sperm proteins that have been implicated in fertilization, those belonging to the ADAM (a disintegrin and metalloprotease) family of proteases have received considerable attention. The focus of the studies described herein has been the characterization of a closely related member of this protease family, ADAMTS10 (a disintegrin and metalloprotease with thrombospondin type 1 motifs number 10). We have demonstrated that ADAMTS10 is expressed during the later stages of mouse spermatogenesis and incorporated into the acrosomal domain of developing spermatids. During sperm maturation, the protein appears to be processed before being expressed on the surface of the peri-acrosomal region of the head. Our collective data suggest that, from this position, ADAMTS10 participates in sperm adhesion to the zona pellucida. Indeed, pre-incubation of capacitated spermatozoa with either galardin, a broad spectrum inhibitor of metalloprotease activity, or anti-ADAMTS10 antisera elicited a significant reduction in their ability to engage in zona adhesion. Overall, these studies support the notion that sperm-oocyte interactions involve considerable functional redundancy and identify ADAMTS10 as a novel candidate in the mediation of these fundamentally important events.

The Simmet lecture: new horizons on an old landscape--oxidative stress, DNA damage and apoptosis in the male germ line.

Our ability to diagnose and treat male infertility is gradually improving in concert with advances in our understanding of the molecular mechanisms underpinning defective sperm function. In this context, one of the factors to emerge as a major causative agent in male infertility is oxidative stress. Spermatozoa are particularly susceptible to such stress because they are exceptionally rich in vulnerable substrates such as polyunsaturated fatty acids, proteins and DNA. The lack of sperm cytoplasm also provides these cells with little capacity to protect themselves from oxidative attack or to effect any repair, should damage occur. Similarly, sperm chromatin is in a quasi-crystalline state and has very little capacity to respond to any DNA damage induced by oxidative attack. When the latter does occur, it appears to be initiated by reactive oxygen species (ROS) generated by the sperm mitochondria. These free radicals attack the lipids present in the sperm mitochondria generating electrophilic aldehydes, which bind to components of the mitochondrial electron transport chain stimulating yet more ROS production. The oxidative stress created via this self-propagating mechanism initiates an apoptotic cascade as a result of which the spermatozoa loose their capacity for fertilization and suffer damage to their DNA. Phosphatidylserine externalization is a late event in sperm apoptosis and may facilitate the silent phagocytosis of moribund cells in the female reproductive tract, that is, the phagocytosis of senescent spermatozoa without the accompanying generation of an inflammatory response. Encouragingly, the involvement of oxidative stress in the aetiology of male infertility has opened up new opportunities for therapeutic interventions involving the judicious administration of nucleophiles and other forms of antioxidants.

An increased frequency of NK cell receptor and HLA-C group 1 combinations in early-onset type 1 diabetes.

AIMS/HYPOTHESIS: Natural killer (NK) cells serve as primary immune surveillance and are partially regulated by combinations of killer immunoglobulin-like receptor (KIR) genes and their HLA class I ligands. Alterations in NK cell activity have been associated with type 1 diabetes. The aim of this study was to determine whether KIR-HLA class I gene frequency: (1) is altered in a current population with type 1 diabetes compared with healthy controls; and (2) has changed over the half century in which the incidence of type 1 diabetes has increased rapidly. METHODS: KIR-HLA class I gene frequencies were compared in 551 individuals diagnosed with type 1 diabetes ≤ 15 years of age (394 in a current cohort and 157 from the historical 'Golden Years' cohort) and 168 healthy controls. The overall balance of activation and inhibition was analysed using KIR-HLA genotype models. RESULTS: Children with type 1 diabetes who were positive for KIR2DS2/KIR2DL2 and KIR2DL3 were more often homozygous for HLA-C group 1 and this effect was strongest in children diagnosed with diabetes before the age of 5 years (p = 0.003, corrected p [p (corr)] = 0.012) and (p = 0.001, p (corr) = 0.004), respectively. Children with type 1 diabetes have fewer inhibitory KIRs with their corresponding ligands compared with healthy controls (p = 1.9 × 10(-4)). This pattern of NK activation has not changed significantly in individuals with type 1 diabetes over the last half century. CONCLUSIONS/INTERPRETATION: Activating combinations of KIR-HLA genes are more frequent in young children with type 1 diabetes diagnosed in the first 5 years of life, suggesting that NK cell responses may be altered in this group.

The electrophoretic separation of spermatozoa: an analysis of genotype, surface carbohydrate composition and potential for capacitation.

This study examines the properties of an electrophoretic device designed to effect the rapid isolation of spermatozoa for assisted conception purposes. In light of previous reports suggesting that X- and Y-bearing spermatozoa can be separated in an electric field, the first characteristic examined was the sex chromosome status of electrophoretically isolated spermatozoa. Exploiting sex chromosome-specific differences in the structure of the amelogenin gene, a quantitative PCR protocol was designed that allowed the rapid genotyping of isolated sperm suspensions. Reassuringly, application of this procedure demonstrated that the electrophoretic method did not result in a significant skewing of the ratio of X- and Y-bearing spermatozoa. Analysis of the molecular basis for electrophoretic sperm isolation demonstrated that sperm suspensions prepared in this manner were enriched in surface sialic acid residues that bound the Sambucus nigra agglutinin (SNA) lectin. Western blot analyses demonstrated the presence of four major SNA binding proteins, three of which were identified by MALDI-Tof mass spectrometry as aminopeptidase B, fucosyltransferase and prostatic acid phosphatase. The ability of neuraminidase to significantly suppress the electrophoretic isolation of spermatozoa emphasized the causative nature of this association between cell surface sialation and sperm behaviour in an electric field. Finally, seminal plasma proteins possessing decapacitation properties were shown to co-migrate with spermatozoa during their electrophoresis, necessitating their removal prior to in vitro fertilization. In terms of function, electrophoretically isolated cells were found to capacitate normally, exhibiting high levels of tyrosine phosphorylation and a capacity for extensive binding to homologous zonae pellucidae. We conclude that the electrophoretic procedure rapidly isolates functional spermatozoa via mechanisms that are independent of their genotype but reliant upon a net electronegative charge that is largely conferred by sperm surface glycoproteins.

On the possible origins of DNA damage in human spermatozoa.

DNA damage in the male germ line has been linked with a variety of adverse clinical outcomes including impaired fertility, an increased incidence of miscarriage and an enhanced risk of disease in the offspring. The origins of this DNA damage could, in principle, involve: (i) abortive apoptosis initiated post meiotically when the ability to drive this process to completion is in decline (ii) unresolved strand breaks created during spermiogenesis to relieve the torsional stresses associated with chromatin remodelling and (iii) oxidative stress. In this article, we present a two-step hypothesis for the origins of DNA damage in human spermatozoa that highlights the significance of oxidative stress acting on vulnerable, poorly protaminated cells generated as a result of defective spermiogenesis. We further propose that these defective cells are characterized by several hallmarks of 'dysmaturity' including the retention of excess residual cytoplasm, persistent nuclear histones, poor zona binding and disrupted chaperone content. The oxidative stress experienced by these cells may originate from infiltrating leukocytes or, possibly, the entry of spermatozoa into an apoptosis-like cascade characterized by the mitochondrial generation of reactive oxygen species. This oxidative stress may be exacerbated by a decline in local antioxidant protection, particularly during epididymal maturation. Finally, if oxidative stress is a major cause of sperm DNA damage then antioxidants should have an important therapeutic role to play in the clinical management of male infertility. Carefully controlled studies are now needed to critically examine this possibility.