Reactive oxygen species in spermatozoa: methods for monitoring and significance for the origins of genetic disease and infertility

Human spermatozoa generate low levels of reactive oxygen species in order to stimulate key events, such as tyrosine phosphorylation, associated with sperm capacitation. However, if the generation of these potentially pernicious oxygen metabolites becomes elevated for any reason, spermatozoa possess a limited capacity to protect themselves from oxidative stress. As a consequence, exposure of human spermatozoa to intrinsically- or extrinsically- generated reactive oxygen intermediates can result in a state of oxidative stress characterized by peroxidative damage to the sperm plasma membrane and DNA damage to the mitochondrial and nuclear genomes. Oxidative stress in the male germ line is associated with poor fertilization rates, impaired embryonic development, high levels of abortion and increased morbidity in the offspring, including childhood cancer. In this review, we consider the possible origins of oxidative damage to human spermatozoa and reflect on the important contribution such stress might make to the origins of genetic disease in our species

Antioxidant systems and oxidative stress in the testes

Spermatogenesis is an extremely active replicative process capable of generating approximately 1,000 sperm a second. The high rates of cell division inherent in this proccss simply correspondingly high rates of mitochondrial oxygen consumption by the germinal epirheliun. However, the poor vascularizarion of the testes means that oxygen tensions in this tissue are low and that competition for this vital element within the testes is extremely intense. Since both spermatogenesis and Leydig cell steroidogenesis are vunerable to oxidative stress, the low oxygen tension that characterizes this tissue may be an important component of the mechanisms by which the testes protects itself from free radical-mediated damage. In addition, the testes contain an elaborate array of antioxidant enzymes and free radical scavengers to ensure that the twin spermatogenic and steroidogenic functions of this organare are not impacted by oxidative stress. These antioxidant defence systems are of major importance because peroxidative damage is currently regarded as the single most important cause of imnpaired testicular function underpinning the pathological consequences of a wide range of conditions from testicular torsion to diabetes and xenobiotic exposure. This chapter sets out the specific nature of these antioxidant defence systems and also reviews the factors that have been found to impair their activity, precipitating a state of oxidative stress in the testes and impairing thetlatter's ability to produce viable spermatozoa capable of initiating and supporting embryonic development

Data on the concentrations of etoposide, PSC833, BAPTA-AM, and cycloheximide that do not compromise the vitality of mature mouse oocytes, parthenogenetically activated and fertilized embryos

AbstractThese data document the vitality of mature mouse oocytes (Metaphase II (MII)) and early stage embryos (zygotes) following exposure to the genotoxic chemotherapeutic agent, etoposide, in combination with PSC833, a selective inhibitor of permeability glycoprotein. They also illustrate the vitality of parthenogenetically activated and fertilized embryos following incubation with the calcium chelator BAPTA-AM (1,2-Bis(2-aminophenoxy)ethane- N,N,N′,N′-tetraacetic acid tetrakis (acetoxymethyl ester)), cycloheximide (an antibiotic that is capable of inhibiting protein synthesis), and hydrogen peroxide (a potent reactive oxygen species). Finally, they present evidence that permeability glycoprotein is not represented in the proteome of mouse spermatozoa. Our interpretation and discussion of these data feature in the article “Identification of a key role for permeability glycoprotein in enhancing the cellular defense mechanisms of fertilized oocytes” (Martin et al., in press) [1]

Analysis of the mechanism by which calcium negatively regulates the tyrosine phosphorylation cascade associated with sperm capacitation

The capacitation of mammalian spermatozoa involves the activation of a cAMP-mediated signal transduction pathway that drives tyrosine phosphorylation via mechanisms that are unique to this cell type. Controversy surrounds the impact of extracellular calcium on this process, with positive and negative effects being recorded in independent publications. We clearly demonstrate that the presence of calcium in the external medium decreases tyrosine phosphorylation in both human and mouse spermatozoa. Under these conditions, a rise in intracellular pH was recorded, however, this event was not responsible for the observed changes in phosphotyrosine expression. Rather, the impact of calcium on tyrosine phosphorylation in these cells was associated with an unexpected change in the intracellular availability of ATP. Thus, the ATP content of both human and mouse spermatozoa fell significantly when these cells were incubated in the presence of external calcium. Furthermore, the removal of glucose, or addition of 2-deoxyglucose, decreased ATP levels within human spermatozoon populations and induced a corresponding decline in phosphotyrosine expression. In contrast, the mitochondrial inhibitor rotenone had no effect on either ATP levels or tyrosine phosphorylation. Addition of the affinity-labeling probe 8-N3 ATP confirmed our prediction that spermatozoa have many calcium-dependent ATPases. Moreover, addition of the ATPase inhibitor thapsigargin, increased intracellular calcium levels, decreased ATP and suppressed tyrosine phosphorylation. Based on these findings, the present study indicates that extracellular calcium suppresses tyrosine phosphorylation by decreasing the availability of intracellular ATP, and not by activating tyrosine phosphatases or inhibiting tyrosine kinases as has been previously suggested.Mark A. Baker, Louise Hetherington, Heath Ecroyd, Shaun D. Roman, and R. John Aitke

Mobile Phone Radiation Induces Reactive Oxygen Species Production and DNA Damage in Human Spermatozoa In Vitro

Background: In recent times there has been some controversy over the impact of electromagnetic radiation on human health. The significance of mobile phone radiation on male reproduction is a key element of this debate since several studies have suggested a relationship between mobile phone use and semen quality. The potential mechanisms involved have not been established, however, human spermatozoa are known to be particularly vulnerable to oxidative stress by virtue of the abundant availability of substrates for free radical attack and the lack of cytoplasmic space to accommodate antioxidant enzymes. Moreover, the induction of oxidative stress in these cells not only perturbs their capacity for fertilization but also contributes to sperm DNA damage. The latter has, in turn, been linked with poor fertility, an increased incidence of miscarriage and morbidity in the offspring, including childhood cancer. In light of these associations, we have analyzed the influence of RF-EMR on the cell biology of human spermatozoa in vitro. Principal Findings: Purified human spermatozoa were exposed to radio-frequency electromagnetic radiation (RF-EMR) tuned to 1.8 GHz and covering a range of specific absorption rates (SAR) from 0.4 W/kg to 27.5 W/kg. In step with increasing SAR, motility and vitality were significantly reduced after RF-EMR exposure, while the mitochondrial generation of reactive oxygen species and DNA fragmentation were significantly elevated (P<0.001). Furthermore, we also observed highly significant relationships between SAR, the oxidative DNA damage bio-marker, 8-OH-dG, and DNA fragmentation after RF-EMRexposure. Conclusions: RF-EMR in both the power density and frequency range of mobile phones enhances mitochondrial reactive oxygen species generation by human spermatozoa, decreasing the motility and vitality of these cells while stimulating DNA base adduct formation and, ultimately DNA fragmentation. These findings have clear implications for the safety of extensive mobile phone use by males of reproductive age, potentially affecting both their fertility and the health and wellbeing of their offspring

The Neon Abundance of Galactic Wolf-Rayet Stars

The fast, dense winds which characterize Wolf-Rayet (WR) stars obscure their underlying cores, and complicate the verification of evolving core and nucleosynthesis models. Core evolution can be probed by measuring abundances of wind-borne nuclear processed elements, partially overcoming this limitation. Using ground-based mid-infrared spectroscopy and the 12.81um [NeII] emission line measured in four Galactic WR stars, we estimate neon abundances and compare to long-standing predictions from evolved-core models. For the WC star WR121, this abundance is found to be >~11x the cosmic value, in good agreement with predictions. For the three less-evolved WN stars, little neon enhancement above cosmic values is measured, as expected. We discuss the impact of clumping in WR winds on this measurement, and the promise of using metal abundance ratios to eliminate sensitivity to wind density and ionization structure.Comment: Accepted for publication in ApJ; 9 pages, 2 color figures, 4 table

Accurate Wavenumbers for Mid-Infrared Fine-Structure Lines

We present accurate new wavenumbers for a set of 13 mid-infrared fine-structure lines. The wavenumbers were determined from observations of the planetary nebula NGC 7027 and of the red supergiant Alpha Scorpii. Most of the new wavenumbers are good to within 0.0025%, or 8 km/s. We provide details on the measurements and present an analysis of the errors. In addition, we present the first observations of hyperfine splitting in the [Na IV] 1106 cm-1 line.Comment: 12 pages text, 2 postscript figures, uses AASTeX macros, figures are gzipped and uuencode