For a long time, superoxide generation by an NADPH oxidase was considered as an oddity only found in professional phagocytes. Over the last years, six homologs of the cytochrome subunit of the phag...

/pdf/the-nox-family-of-ros-generating-nadph-oxidases-physiology-zjc88zvh59.pdf

The NOX Family of ROS-Generating NADPH Oxidases: Physiology and Pathophysiology

https://www.commed.vcu.edu/Chronic_Disease/2012/cochranrvwrschbias.pdf

The Cochrane Collaboration's tool for assessing risk of bias in randomised trials

WHO laboratory manual for the examination and processing of human semen

Tetrazolium salts have become some of the most widely used tools in cell biology for measuring the metabolic activity of cells ranging from mammalian to microbial origin. With mammalian cells, fractionation studies indicate that the reduced pyridine nucleotide cofactor, NADH, is responsible for most MTT reduction and this is supported by studies with whole cells. MTT reduction is associated not only with mitochondria, but also with the cytoplasm and with non-mitochondrial membranes including the endosome/lysosome compartment and the plasma membrane. The net positive charge on tetrazolium salts like MTT and NBT appears to be the predominant factor involved in their cellular uptake via the plasma membrane potential. However, second generation tetrazolium dyes that form water-soluble formazans and require an intermediate electron acceptor for reduction (XTT, WST-1 and to some extent, MTS), are characterised by a net negative charge and are therefore largely cell-impermeable. Considerable evidence indicates that their reduction occurs at the cell surface, or at the level of the plasma membrane via trans-plasma membrane electron transport. The implications of these new findings are discussed in terms of the use of tetrazolium dyes as indicators of cell metabolism and their applications in cell biology.

Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction.

Despite the skepticism that once prevailed among immunologists, it is now widely accepted that the normal immune system harbors a T-cell population, called regulatory T cells (Treg cells), specialized for immune suppression. It was first shown that depletion of a T-cell subpopulation from normal rodents produced autoimmune disease. Search for a molecular marker specific for such autoimmune-preventive Treg cells has revealed that the majority, if not all, of them constitutively express the CD25 molecule as depletion of CD25+CD4+ T cells spontaneously evokes autoimmune disease in otherwise normal rodents. The expression of CD25 by Treg cells has made it possible to delineate their developmental pathways, in particular their thymic development, and establish simple in vitro assay for assessing their suppressive activity. The marker and the in vitro assay have helped to identify human Treg cells with similar functional and phenotypic characteristics. Recent efforts have shown that natural Treg cells specifically express the transcription factor Foxp3 and that mutations of the Foxp3 gene produce a variety of immunological diseases in humans and rodents. Specific expression of Foxp3 in natural Treg cells has enabled their functional and developmental characterization by genetic approach. These studies altogether have provided firm evidence for Foxp3+CD25+CD4+ Treg cells as an indispensable cellular constituent of the normal immune system for establishing and maintaining immunologic self-tolerance and immune homeostasis. Treg cells are now within the scope of clinical use to treat immunological diseases and control physiological and pathological immune responses.

Regulatory T Cells

Recent studies have demonstrated that human spermatozoa are capable of generating reactive oxygen species and that this activity is significantly accelerated in cases of defective sperm function. In view of the pivotal role played by lipid peroxidation in mediating free radical damage to cells, we have examined the relationships between reactive oxygen species production, lipid peroxidation, and the functional competence of human spermatozoa. Using malondialdehyde production in the presence of ferrous ion promoter as an index of lipid peroxidation, we have shown that lipid peroxidation is significantly accelerated in populations of defective spermatozoa exhibiting high levels of reactive oxygen species production or in normal cells stimulated to produce oxygen radicals by the ionophore, A23187. The functional consequences of lipid peroxidation included a dose-dependent reduction in the ability of human spermatozoa to exhibit sperm oocyte-fusion, which could be reversed by the inclusion of a chain-breaking antioxidant, alpha-tocopherol. Low levels of lipid peroxidation also had a slight enhancing effect on the generation of reactive oxygen species in response to ionophore, without influencing the steady-state activity. At higher levels of lipid peroxidation, both the basal level of reactive oxygen species production and the response to A23187 were significantly diminished. In contrast, lipid peroxidation had a highly significant, enhancing effect on the ability of human spermatozoa to bind to both homologous and heterologous zonae pellucidae via mechanisms that could again be reversed by alpha-tocopherol. These results are consistent with a causative role for lipid peroxidation in the etiology of defective sperm function and also suggest a possible physiological role for the reactive oxygen species generated by human spermatozoa in mediating sperm-zona interaction.

Generation of reactive oxygen species, lipid peroxidation, and human sperm function.

Addition of the divalent cation ionophore, A23187, to washed populations of human spermatozoa resulted in a sudden burst of production of reactive oxygen species which peaked within 3-5 min. This activity was dependent upon the presence of calcium in the external medium and was unaffected by the mitochondrial inhibitors, oligomycin, antimycin and rotenone. Studies with scavengers of reactive oxygen species revealed that, while reagents directed against singlet oxygen and the hydroxyl radical were without effect, cytochrome C reduced the response to A23187 by about 50%, suggesting that the superoxide anion radical is a major product of the activated human spermatozoon. The clinical implications of these studies stem from the considerable variation observed between individuals in the levels of reactive oxygen species produced by the spermatozoa. This variability was shown to be inversely related to the ability of the spermatozoa to exhibit sperm-oocyte fusion on exposure to A23187; defective samples exhibited a basal level of reactive oxygen species production which was 40 times that observed with normal functional cells.

/pdf/cellular-basis-of-defective-sperm-function-and-its-zab3dedi9x.pdf

Cellular basis of defective sperm function and its association with the genesis of reactive oxygen species by human spermatozoa.

Reactive oxygen metabolites are known to disrupt sperm-oocyte fusion, sperm movement, and DNA integrity; however, the relative sensitivities of these elements to oxidative stress are unknown. In this study these factors were assessed in human spermatozoa exposed to increasing levels of oxidative stress achieved through the stimulation of endogenous oxidant generation with NADPH or direct exposure to hydrogen peroxide. At low levels of oxidative stress, DNA fragmentation was significantly reduced while the rates of sperm-oocyte fusion were significantly enhanced. As the level of oxidative stress increased, the spermatozoa exhibited significantly elevated levels of DNA damage (p < 0.001) and yet continued to express an enhanced capacity for sperm-oocyte fusion. At the highest levels of oxidative stress, extremely high rates of DNA fragmentation were observed but the spermatozoa exhibited a parallel loss in their capacities for movement and oocyte fusion. These studies emphasize how redox mechanisms can either enhance or disrupt the functional and genomic integrity of human spermatozoa depending on the intensity of the oxidative stimulus. Because these qualities are affected at different rates, spermatozoa exhibiting significant DNA damage are still capable of fertilizing the oocyte. These results may have long-term implications for the safety of assisted conception procedures in cases associated with oxidative stress.

/pdf/relative-impact-of-oxidative-stress-on-the-functional-57r02fvx60.pdf

Relative Impact of Oxidative Stress on the Functional Competence and Genomic Integrity of Human Spermatozoa

Recent advances in understanding of male infertility have implicated two major causative factors, oxidative stress and Y chromosome deletions. A major cause of oxidative stress appears to be the high rate of reactive oxygen species generation associated with the retention of excess residual cytoplasm in the sperm midpiece. Other possible causes include the redox cycling of xenobiotics, and antioxidant depletion or apoptosis. Oxidative stress induces peroxidative damage in the sperm plasma membrane and DNA damage in both the mitochondrial and nuclear genomes. Nuclear DNA damage in the germ line of the father may be associated with pathology in the offspring, including childhood cancer and infertility. Gene deletions on the non-recombining region of the Y chromosome account for the infertility observed in about 15% of patients with azoospermia and 5-10% of subjects with severe oligozoospermia. The Y chromosome is particularly susceptible to gene deletions because of the inability of the haploid genome to deploy recombination repair in retrieving lost genetic information. Aberrant recombination, defective chromatin packaging, abortive apoptosis and oxidative stress may all be involved in the aetiology of DNA damage in the germ line. The factors responsible for Y chromosome deletions in spermatozoa remain unresolved but may be one facet of a central reproductive problem: controlling the amount of oxidative stress experienced by germ cells during their differentiation and maturation in the male reproductive tract.

/pdf/oxidative-stress-dna-damage-and-the-y-chromosome-3dr9oq8vcx.pdf

Oxidative stress, DNA damage and the Y chromosome.

The literature contains conflicting evidence regarding the existence of DNA damage in spermatozoa from infertile male patients. To examine this phenomenon, we have studied ejaculated spermatozoa from normozoospermic semen donors and from a group of the unselected male partners of couples attending an infertility clinic for initial investigation. Classical semen analysis according to World Health Organization (WHO) guidelines was undertaken with computer-assisted sperm analysis (CASA). Spermatozoa were prepared by sequential washing and centrifugation and were analyzed for DNA fragmentation using three assays: 1) a single-cell gel electrophoresis (comet) assay, 2) in situ nick translation with prior chemical decondensation (ISNT-decondensed), and 3) in situ nick translation without prior chemical decondensation (ISNT-condensed). In addition, reactive oxygen species (ROS) generation by spermatozoa was measured, and seminal plasma was analyzed for its total reactive antioxidant potential (TRAP). When the donor and patient groups were compared, the latter had lower levels of semen quality and higher levels of DNA damage, which was particularly apparent using the comet assay. Highly significant negative correlations were observed between DNA fragmentation, detected by all three assays, and semen quality, particularly sperm concentration. In addition, multiple regression analysis indicated that other attributes of semen quality, such as sperm movement and ROS generation, were also related to DNA damage. We conclude that a significant proportion of infertile men have elevated levels of DNA damage in their ejaculated spermatozoa.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/j.1939-4640.2000.tb03273.x

Robert John Aitken

Papers

Generation of reactive oxygen species, lipid peroxidation, and human sperm function.

Cellular basis of defective sperm function and its association with the genesis of reactive oxygen species by human spermatozoa.

Relative Impact of Oxidative Stress on the Functional Competence and Genomic Integrity of Human Spermatozoa

Oxidative stress, DNA damage and the Y chromosome.

DNA Integrity in Human Spermatozoa: Relationships With Semen Quality