One of the major causes of defective sperm function is oxidative stress, which not only disrupts the integrity of sperm DNA but also limits the fertilizing potential of these cells as a result of ...collateral damage to proteins and lipids in the sperm plasma membrane. The origins of such oxidative stress appear to involve the sperm mitochondria, which have a tendency to generate high leve｜s of superoxide anion as a prelude to entering the intrinsic apoptotic cascade. Unfortunately, these cells have very little capacity to respond to such an attack because they only possess the first enzyme in the base excision repair （BER） pathway, 8-oxoguanine glycosylase 1 （OGG1）. The latter successfully creates an abasic site, but the spermatozoa cannot process the oxidative lesion further because they lack the downstream proteins （APE1, XRCC1） needed to complete the repair process. It is the responsibility of the oocyte to continue the BER pathway prior to initiation of S-phase of the first mitotic division. If a mistake is made by the oocyte at this stage of development, a mutation will be created that will be represented in every cell in the body. Such mechanisms may explain the increase in childhood cancers and other diseases observed in the offspring of males who have suffered oxidative stress in their germ line as a consequence of age, environmental or lifestyle factors. The high prevalence of oxidative DNA damage in the spermatozoa of male infertility patients may have implications for the health of children conceived in vitro and serves as a driver for current research into the origins of free radical generation in the germ line.
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.
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-EMR exposure.
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 reproductive consequences of global warming are not currently understood. In order to address this issue, we have examined the reproductive consequences of exposingmalemice to a mild heat stress. ...For this purpose, adultmalemice were exposed to an elevated ambient temperature of 35°C under two exposure models. The first involved acute exposure for 24 h, followed by recovery periods between 1 day and 6 weeks. The alternative heating regimen involved a daily exposure of 8 h for periods of 1 or 2 weeks. In our acute model, we identified elevated sperm mitochondrial ROS generation (P < 0.05), increased sperm membrane fluidity (P < 0.05), DNA damage in the form of single-strand breaks (P<0.001), and oxidative DNAdamage (P<0.05), characteristic of an oxidative stress cascade. This DNA damage was detected in pachytene spermatocytes (P < 0.001) and round spermatids (P < 0.001) isolated from testes after 1 day heat recovery. Despite these lesions, the spermatozoa of heat-treated mice exhibited no differences in their ability to achieve hallmarks of capacitation or to fertilize the oocyte and support development of embryos to the blastocyst stage (all P>0.05). Collectively, our acute heat stress model supports the existence of heat susceptible stages of germ cell development, with the round spermatids being most perturbed and spermatogonial stem cells exhibiting resistance to this insult. Such findings were complemented by our chronic heat stress model, which further supported the vulnerability of the round spermatid population. Summary Sentence Environmental heating induces a state of oxidative stress in the male germ line, affecting multiple germ cell types; primarily the round spermatid and pachytene spermatocyte populations.
Context: Male infertility has been linked with the excessive generation of reactive oxygen species (ROS) by defective spermatozoa. However, the subcellular origins of this activity are unclear.
...Objective: The objective of this study was to determine the importance of sperm mitochondria in creating the oxidative stress associated with defective sperm function.
Method: Intracellular measurement of mitochondrial ROS generation and lipid peroxidation was performed using the fluorescent probes MitoSOX red and BODIPY C11 in conjunction with flow cytometry. Effects on sperm movement were measured by computer-assisted sperm analysis.
Results: Disruption of mitochondrial electron transport flow in human spermatozoa resulted in generation of ROS from complex I (rotenone sensitive) or III (myxothiazol, antimycin A sensitive) via mechanisms that were independent of mitochondrial membrane potential. Activation of ROS generation at complex III led to the rapid release of hydrogen peroxide into the extracellular space, but no detectable peroxidative damage. Conversely, the induction of ROS on the matrix side of the inner mitochondrial membrane at complex I resulted in peroxidative damage to the midpiece and a loss of sperm movement that could be prevented by the concomitant presence of α-tocopherol. Defective human spermatozoa spontaneously generated mitochondrial ROS in a manner that was negatively correlated with motility. Simultaneous measurement of general cellular ROS generation with dihydroethidium indicated that 68% of the variability in such measurements could be explained by differences in mitochondrial ROS production.
Conclusion: We conclude that the sperm mitochondria make a significant contribution to the oxidative stress experienced by defective human spermatozoa.
Diffuse midline glioma (DMG) is a deadly pediatric and adolescent central nervous system (CNS) tumor localized along the midline structures of the brain atop the spinal cord. With a median overall ...survival (OS) of just 9-11-months, DMG is characterized by global hypomethylation of histone H3 at lysine 27 (H3K27me3), driven by recurring somatic mutations in H3 genes including, HIST1H3B/C (H3.1K27M) or H3F3A (H3.3K27M), or through overexpression of EZHIP in patients harboring wildtype H3. The recent World Health Organization's 5th Classification of CNS Tumors now designates DMG as, 'H3 K27-altered', suggesting that global H3K27me3 hypomethylation is a ubiquitous feature of DMG and drives devastating transcriptional programs for which there are no treatments. H3-alterations co-segregate with various other somatic driver mutations, highlighting the high-level of intertumoral heterogeneity of DMG. Furthermore, DMG is also characterized by very high-level intratumoral diversity with tumors harboring multiple subclones within each primary tumor. Each subclone contains their own combinations of driver and passenger lesions that continually evolve, making precision-based medicine challenging to successful execute. Whilst the intertumoral heterogeneity of DMG has been extensively investigated, this is yet to translate to an increase in patient survival. Conversely, our understanding of the non-genomic factors that drive the rapid growth and fatal nature of DMG, including endogenous and exogenous microenvironmental influences, neurological cues, and the posttranscriptional and posttranslational architecture of DMG remains enigmatic or at best, immature. However, these factors are likely to play a significant role in the complex biological sequelae that drives the disease. Here we summarize the heterogeneity of DMG and emphasize how analysis of the posttranslational architecture may improve treatment paradigms. We describe factors that contribute to treatment response and disease progression, as well as highlight the potential for pharmaco-proteogenomics (i.e., the integration of genomics, proteomics and pharmacology) in the management of this uniformly fatal cancer.
The testicular spermatozoa of all mammalian species are considered functionally immature owing to their inability to swim in a progressive manner and engage in productive interactions with the ...cumulus-oocyte complex. The ability to express these key functional attributes develops progressively during the cells' descent through the epididymis, a highly specialized ductal system that forms an integral part of the male reproductive tract. The functional maturation of the spermatozoon is achieved
continuous interactions with the epididymal luminal microenvironment and remarkably, occurs in the complete absence of
gene transcription or protein translation. Compositional analysis of the luminal fluids collected from the epididymis of a variety of species has revealed the complexity of this milieu, with a diversity of inorganic ions, proteins, and small non-coding RNA transcripts having been identified to date. Notably, both the quantitative and qualitative profile of each of these different luminal elements display substantial segment-to-segment variation, which in turn contribute to the regionalized functionality of this long tubule. Thus, spermatozoa acquire functional maturity in the proximal segments before being stored in a quiescent state in the distal segment in preparation for ejaculation. Such marked division of labor is achieved
the combined secretory and absorptive activity of the epithelial cells lining each segment. Here, we review our current understanding of the molecular mechanisms that exert influence over the unique intraluminal environment of the epididymis, with a particular focus on vesicle-dependent mechanisms that facilitate intercellular communication between the epididymal soma and maturing sperm cell population.
Acute myeloid leukaemia (AML) is an aggressive haematological malignancy with a poor overall survival. Reactive oxygen species (ROS) have been shown to be elevated in a wide range of cancers ...including AML. Whilst previously thought to be mere by-products of cellular metabolism, it is now clear that ROS modulate the function of signalling proteins through oxidation of critical cysteine residues. In this way, ROS have been shown to regulate normal haematopoiesis as well as promote leukaemogenesis in AML. In addition, ROS promote genomic instability by damaging DNA, which promotes chemotherapy resistance. The source of ROS in AML appears to be derived from members of the "NOX family" of NADPH oxidases. Most studies link NOX-derived ROS to activating mutations in the Fms-like tyrosine kinase 3 (FLT3) and Ras-related C3 botulinum toxin substrate (Ras). Targeting ROS through either ROS induction or ROS inhibition provides a novel therapeutic target in AML. In this review, we summarise the role of ROS in normal haematopoiesis and in AML. We also explore the current treatments that modulate ROS levels in AML and discuss emerging drug targets based on pre-clinical work.
Spermatogonial stem cells (SSCs) are generally characterized by excellent DNA surveillance and repair, resulting in one of the lowest spontaneous mutation rates in the body. However, the barriers to ...mutagenesis can be overwhelmed under two sets of circumstances. First, replication errors may generate age-dependent mutations that provide the mutant cells with a selective advantage, leading to the clonal expansions responsible for dominant genetic diseases such as Apert syndrome and achondroplasia. The second mechanism centers on the vulnerability of the male germline to oxidative stress and the induction of oxidative DNA damage in spermatozoa. Defective repair of such oxidative damage in the fertilized oocyte results in the creation of mutations in the zygote that can influence the health and well-being of the offspring. A particular hot spot for such oxidative attack on chromosome 15 has been found to align with several mutations responsible for paternally mediated disease, including cancer, psychiatric disorders, and infertility.
An increase in intraovarian reactive oxygen species (ROS) has long been implicated in the decline in oocyte quality associated with maternal ageing. Oxidative stress (OS)-induced lipid peroxidation ...and the consequent generation of highly electrophilic aldehydes, such as 4-hydroxynonenal (4-HNE), represents a potential mechanism by which ROS can inflict damage in the ageing oocyte. In this study, we have established that aged oocytes are vulnerable to damage by 4-HNE resulting from increased cytosolic ROS production within the oocyte itself. Further, we demonstrated that the age-related induction of OS can be recapitulated by exposure of germinal vesicle (GV) oocytes to exogenous H
O
. Such treatments stimulated an increase in 4-HNE generation, which remained elevated during in vitro oocyte maturation to metaphase II. Additionally, exposure of GV oocytes to either H
O
or 4-HNE resulted in decreased meiotic completion, increased spindle abnormalities, chromosome misalignments and aneuploidy. In seeking to account for these data, we revealed that proteins essential for oocyte health and meiotic development, namely α-, β-, and γ-tubulin are vulnerable to adduction via 4-HNE. Importantly, 4-HNE-tubulin adduction, as well as increased aneuploidy rates, were resolved by co-treatment with the antioxidant penicillamine, demonstrating a possible therapeutic mechanism to improve oocyte quality in older females.
The prolonged incubation of human spermatozoa in vitro was found to induce a loss of motility associated with the activation of mitochondrial reactive oxygen species generation in the absence of any ...change in mitochondrial membrane potential. The increase in mitochondrial free radical production was paralleled by a loss of protein thiols and a concomitant rise in the formation of 4-hydroxynonenal, an electrophilic product of lipid peroxidation that was found to directly suppress sperm movement. These results prompted a search for nucleophiles that could counteract the action of such cytotoxic aldehydes, as a means of ensuring the long-term survival of spermatozoa in vitro. Four nucleophilic compounds were consequently assessed (penicillamine, homocysteine, N-acetylcysteine, and mercaptosuccinate) in three species (human, rat, and horse). The results of this analysis revealed drug and species specificity in the manner in which these compounds affected sperm function, with penicillamine conferring the most consistent, effective support. This prosurvival effect was achieved downstream of mitochondrial reactive oxygen species generation and was associated with the stabilization of 4-hydroxynonenal generation, the preservation of sperm thiols, and a reduction in 8-hydroxy-2'-deoxyguanosine formation. Theoretical calculations of Fe-S and Cu-S bond distances and corresponding binding energies suggested that the particular effectiveness of penicillamine may, in part, reflect the ability of this nucleophile to form stable complexes with transition metals that catalyze lipid peroxidation. The practical implications of these findings were indicated by the effective preservation of equine spermatozoa for 8 days at ambient temperature when the culture medium was supplemented with penicillamine.
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