While the pathological impact of reactive oxygen species (ROS) in the aetiology of human infertility has received much attention, this review explores the counterproposal that these highly reactive metabolites play a positive role in mediating reproductive success. The physiological importance of ROS in biological systems can be distilled into three main categories of influence: (1) ROS can oxidize thiols to generate either the corresponding sulfenic acid or disulfide bridges. This oxidizing capacity is critical for several reproductive processes, including the cross linking of sperm chromatin during epididymal maturation, formation of the mitochondrial sheath, and the activation of proteolytic zymogens involved in such processes as ovulation, menstruation, implantation, and parturition. Thiol oxidation is also involved in the suppression of phosphatase activity and the resulting promotion of phosphorylation-dependent signal transduction pathways, which are involved in virtually every aspect of reproduction from sperm capacitation to parturition; (2) The destructive properties of ROS are also biologically significant in the defence against genital tract infections and in mediating such processes as autophagy, apoptosis, and ferroptosis, which are fundamental to the reproductive process; (3) Finally, ROS are involved in controlling the redox status of transition metals (particularly iron and copper) in the active site of many enzymes that are of fundamental importance to reproduction. Given the biological importance of ROS to procreation, we should use antioxidants with care in managing both male and female infertility and avoid the induction of reductive stress.

Exposure to systemic heat stress in male mammals adversely affects sperm production, fertility, and DNA integrity. To date, few studies have investigated this phenomenon in horses, particularly in industry-relevant environments. Therefore, this study examined the relationship between ambient climatic conditions and fertility within a population of commercially fertile stallions. Post-coital semen samples (n = 804) were collected weekly from 46 Thoroughbred stallions during two successive breeding seasons (NSW, Australia; 22 weeks total). Semen samples were processed via single-layer colloidal centrifugation on-site to remove contaminants, seminal plasma and poor-quality sperm cells. The remaining sperm fraction was resuspended in Biggers, Whitten and Whittingham (BWW) medium for analyses, including sperm concentration, motility, and DNA damage. Fertility data (first cycle and per-cycle pregnancy rates) were collected from farms. Loggers were placed in the stables and paddocks of stallions to record ambient temperature and humidity, on 3-min cycles. Our results indicate that current management regimens, involving the stabling of stallions at night, with paddock access during the day, expose stallions to highest ambient climatic conditions. Polynomial distributed lag modelling identified 18 stallions whose fertility was adversely affected by heat stress, based on correlations between fertility and ambient climatic conditions (p ≤ 0.05). Of these 18 candidate stallions, six (13

Widespread exposure to per- and polyfluoroalkyl substances (PFAS) poses substantial health risks to humans and animals. PFAS have the propensity to bioaccumulate in organs such as the testes, wherein they have been implicated in adverse effects on sperm production and quality. Here, we sought to understand the multigenerational implications of such effects by chronically subjecting three generations of mice to an environmentally relevant PFAS cocktail and subsequently investigating impacts on reproduction, behavior and offspring development. This strategy confirmed that testicular accumulation of PFAS is correlated with compromised rates of sperm production and alterations to the sperm epigenome but did not compromise the overall fertility or behavioral outcomes explored in PFAS-exposed males. Notably, the negative effects were more pronounced in F1 offspring compared with subsequent F2 and F3 generations. These data support mounting evidence of adverse associations between PFAS exposure and reproductive capacity, but suggest the risk posed by PFAS is not amplified across three generations.

Over the past half-century, the world has witnessed a dramatic decline in human fertility, which began in the 1960s and has continued unabated ever since [...]

Exposure to anthropogenic electromagnetic fields (EMFs), especially those of wireless communications (WC) has increased tremendously. This is an unprecedented phenomenon throughout biological evolution because, all anthropogenic EMFs, being fully polarized, coherent, and, especially WC EMFs, highly variable, differ substantially from the natural EMFs. WC EMFs consist of Microwave (MW) carrier waves, modulated, by Extremely Low Frequency (ELF) signals, and included in on/off pulses repeated at various ELF rates. Moreover, they exhibit intense random variability, mainly in the Ultra Low Frequency (ULF) band. Thus, WC EMFs are a combination of MW and ELF/ULF EMFs. The combination of polarization/coherence and intense low-frequency (ELF/ULF) variability seems to be the key to EMF-bioactivity. Epidemiological and laboratory studies highlight a connection between ELF or WC EMF exposure and cancer, infertility, electro-hypersensitivity, and various other pathologies. Studies also find DNA damage and Oxidative Stress (OS) which explain these pathologies. While man-made EMFs cannot directly ionize molecules, they are capable of doing this indirectly in biological tissue, by triggering the biosynthesis of Reactive Oxygen Species (ROS) which can damage biomolecules, including DNA. The (over)production of ROS and the consequent OS are triggered by irregular gating of Voltage-Gated Ion Channels (VGICs) in the cell membranes as described by the Ion Forced Oscillation (IFO)-VGIC mechanism: Mobile ions within VGICs forced to oscillate by the applied ELF/ULF EMFs exert forces on the voltage sensors of the VGICs, similar to or greater than the forces that physiologically gate those channels, resulting in their irregular gating (dysfunction). Dysfunction of ion channels disrupts intracellular ionic concentrations. This triggers ROS overproduction and OS by the ROS-generating systems/enzymes in the cells, such as the electron transport chain (ETC) in the mitochondria, or the NADPH/NADH oxidases (NOXs), the Nitric Oxide synthases (NOS), etc. The IFO-VGIC mechanism and the consequent OS constitute a comprehensive mechanism that explains all known adverse biological and health effects reported to be induced by anthropogenic EMFs.

Assessing oxidative stress levels in spermatozoa provides crucial information on the health of the cell, while also potentially providing insight into the systemic health of the individual. Indeed, elevated oxidative stress in spermatozoa has been implicated in compromising sperm function and, consequently, is now considered one of the leading mediators of numerous male reproductive pathologies. Here we describe two flow cytometric assays: Dihydroethidium and Mitosox Red, which can be used to detect reactive oxygen species originating from the cytosol and mitochondria, respectively, in both mouse and human spermatozoa. Such tools have utility in studies investigating the impact of reproductive aging, toxicants, and environmental exposures on the male germline. These assays are also beneficial in the context of testing novel pharmaceutical interventions or antioxidant defence therapies aimed at preserving fertility or for use in assisted reproduction technologies.

Oxidative stress, characterized by an imbalance between prooxidants and antioxidants in favor of the former, can lead to cellular damage due to the accumulation of reactive oxygen species (ROS) and reactive nitrogen species (RNS). In spermatozoa, oxidative stress plays a crucial role in signaling pathways associated with capacitation, a process essential for fertilization. However, the limited capacity of spermatozoa to buffer ROS makes them susceptible to oxidative stress. Measurement of oxidative stress in spermatozoa is important as it precedes or directly causes the loss of basic sperm functions-such as motility and the membrane changes required for zona binding and gamete fusion-associated with fertility. This chapter aims to describe protocols for assessing oxidative damage in spermatozoa, encompassing the generation of ROS in mitochondria to the manifestation of oxidative damage in DNA, proteins, and lipids. In addition to the detailed instructions provided within this chapter, we have sought to provide background information on the development and significance of the assays which we most commonly utilize in our laboratory.

Per- and polyfluoroalkyl substances (PFAS) are a large group of persistent synthetic chemicals and ubiquitous environmental contaminants. Mounting evidence demonstrates that PFAS can bioaccumulate and induce adverse health outcomes, including compromising male reproduction. Despite this, the mechanisms by which PFAS elicits these effects remain unclear. Here, we investigate how an environmentally relevant PFAS cocktail impacts the reproductive function of male Swiss CD1 mice. Following twelve weeks of continuous exposure, we collected blood samples for hormone and PFAS quantification and processed reproductive tissues and spermatozoa for histological and functional assessment. PFAS exposure significantly reduced the rate of daily sperm production, likely due to decreased circulating testosterone and dihydrotestosterone. Further, PFAS-exposed spermatozoa displayed marked alterations to their small non-coding RNA profile, which were linked to dysregulation of early-embryonic gene expression. Notably, these changes occured without significant alteration in sperm viability, motility, or the ability to undergo capacitation or support embryonic development. These findings provide new mechanistic insight into how PFAS exposure impacts male reproductive health.

Studying DNA damage in spermatozoa allows researchers fundamental insight into the molecular mechanisms underlying the cellular response of sperm to stressors and provides an opportunity for understanding novel mechanisms for mitigating this type of damage. Furthermore, since compromised genetic integrity is recognized as a leading contributor to infertility phenotypes, reliable methods for assessing DNA damage in populations of spermatozoa are essential in research and clinical laboratories alike. Here, we describe two methods, the sperm chromatin structure assay (SCSA) and alkaline comet assay, designed to assess both single- and double-stranded DNA damage within the spermatozoon.

Forecasted increases in the prevalence and severity of extreme weather events accompanying changes in climatic behavior pose potential risk to the reproductive capacity of humans and animals of ecological and agricultural significance. While several studies have revealed that heat stress induced by challenges such as testicular insulation can elicit a marked negative effect on the male reproductive system, and particularly the production of spermatozoa, less is known about the immediate impact on male reproductive function following subchronic whole-body exposure to elevated ambient temperature. To address this knowledge gap, we exposed unrestrained male mice to heat stress conditions that emulate a heat wave (daily cycle of 8 h at 35 °C followed by 16 h at 25 °C) for a period of 7 d. Neither the testes or epididymides of heat-exposed male mice exhibited evidence of gross histological change, and similarly, spermatozoa of exposed males retained their functionality and ability to support embryonic development. However, the embryos generated from heat-exposed spermatozoa experienced pronounced changes in gene expression linked to acceleration of early embryo development, aberrant blastocyst hatching, and increased fetal:placental weight ratio. Such changes were causally associated with an altered sperm small noncoding RNA (sncRNA) profile, such that these developmental phenotypes were recapitulated by microinjection of wild-type embryos sired by control spermatozoa with RNAs extracted from heat-exposed spermatozoa. Such data highlight that even relatively modest excursions in ambient temperature can affect male reproductive function and identify the sperm sncRNA profile as a particular point of vulnerability to this imposed environmental stress.

Oxidative stress can be induced in the testes by a wide range of factors, including scrotal hyperthermia, varicocele, environmental toxicants, obesity and infection. The clinical consequences of such stress include the induction of genetic damage in the male germ line which may, in turn, have serious implications for the health and wellbeing of the progeny. In order to confirm the transgenerational impact of oxidative stress in the testes, we sought to develop an animal model in which this process could be analysed. Our primary approach to this problem was to induce Sertoli cells (robust, terminally differentiated, tissue-specific testicular cells whose radioresistance indicates significant resistance to oxidative stress) to generate high levels of reactive oxygen species (ROS) within the testes. To achieve this aim, six follicle-stimulating hormone (FSH) peptides were developed and compared for selective targeting to Sertoli cells both in vitro and in vivo. Menadione, a redox-cycling agent, was then conjugated to the most promising FSH candidate using a linker that had been optimised to enable maximum production of ROS in the targeted cells. A TM4 Sertoli cell line co-incubated with the FSH-menadione conjugate in vitro exhibited significantly higher levels of mitochondrial ROS generation (10-fold), lipid peroxidation (2-fold) and oxidative DNA damage (2-fold) than the vehicle control. Additionally, in a proof-of-concept study, ten weeks after a single injection of the FSH-menadione conjugate in vivo, injected male mice were found to exhibit a 1.6 fold increase in DNA double strand breaks and 13-fold increase in oxidative DNA damage to their spermatozoa while still retaining their ability to initiate a pregnancy. We suggest this model could now be used to study the influence of chronic oxidative stress on testicular function with emphasis on the impact of DNA damage in the male germ line on the mutational profile and health of future generations.

Graphical abstract Abstract Poly- and per-fluoroalkyl substances (PFAS) are synthetic environmentally persistent chemicals. Despite the phaseout of specific PFAS, their inherent stability has resulted in ubiquitous and enduring environmental contamination. PFAS bioaccumulation has been reported globally with omnipresence in most populations wherein they have been associated with a range of negative health effects, including strong associations with increased instances of testicular cancer and reductions in overall semen quality. To elucidate the biological basis of such effects, we employed an acute in vitro exposure model in which the spermatozoa of adult male mice were exposed to a cocktail of PFAS chemicals at environmentally relevant concentrations. We hypothesized that direct PFAS treatment of spermatozoa would induce reactive oxygen species generation and compromise the functional profile and DNA integrity of exposed cells. Despite this, post-exposure functional testing revealed that short-term PFAS exposure (3 h) did not elicit a cytotoxic effect, nor did it overtly influence the functional profile, capacitation rate, or the in vitro fertilization ability of spermatozoa. PFAS treatment of spermatozoa did, however, result in a significant delay in the developmental progression of the day 4 pre-implantation embryos produced in vitro . This developmental delay could not be attributed to a loss of sperm DNA integrity, DNA damage, or elevated levels of intracellular reactive oxygen species. When considered together, the results presented here raise the intriguing prospect that spermatozoa exposed to a short-term PFAS exposure period potentially harbor an alternate stress signal that is delivered to the embryo upon fertilization. Lay summary PFAS are synthetic chemicals widely used in non-stick cookware, food packaging, and firefighting foam. Such extensive use has led to concerning levels of environmental contamination and reports of associations with a spectrum of negative health outcomes, including testicular cancer and reduced semen quality. To investigate the effects of PFAS on male reproduction, we incubated mouse sperm in a cocktail of nine PFAS at environmentally relevant concentrations before checking for a range of functional outcomes. This treatment strategy was not toxic to the sperm; it did not kill them or reduce their motility, nor did it affect their fertilization capacity. However, we did observe developmental delays among pre-implantation embryos created using PFAS-treated sperm. Such findings raise the intriguing prospect that PFAS-exposed sperm harbor a form of stress signal that they deliver to the embryo upon fertilization.

Mutations in the type III receptor tyrosine kinase FLT3 are frequent in patients with acute myeloid leukemia (AML) and are associated with a poor prognosis. AML is characterized by the overproduction of reactive oxygen species (ROS), which can induce cysteine oxidation in redox-sensitive signaling proteins. Here, we sought to characterize the specific pathways affected by ROS in AML by assessing oncogenic signaling in primary AML samples. The oxidation or phosphorylation of signaling proteins that mediate growth and proliferation was increased in samples from patient subtypes with FLT3 mutations. These samples also showed increases in the oxidation of proteins in the ROS-producing Rac/NADPH oxidase-2 (NOX2) complex. Inhibition of NOX2 increased the apoptosis of FLT3-mutant AML cells in response to FLT3 inhibitors. NOX2 inhibition also reduced the phosphorylation and cysteine oxidation of FLT3 in patient-derived xenograft mouse models, suggesting that decreased oxidative stress reduces the oncogenic signaling of FLT3. In mice grafted with FLT3 mutant AML cells, treatment with a NOX2 inhibitor reduced the number of circulating cancer cells, and combining FLT3 and NOX2 inhibitors increased survival to a greater extent than either treatment alone. Together, these data raise the possibility that combining NOX2 and FLT3 inhibitors could improve the treatment of FLT3 mutant AML.

Male infertility is a commonly encountered pathology that is estimated to be a contributory factor in approximately 50% of couples seeking recourse to assisted reproductive technologies. Upon clinical presentation, such males are commonly subjected to conventional diagnostic andrological practices that rely on descriptive criteria to define their fertility based on the number of morphologically normal, motile spermatozoa encountered within their ejaculate. Despite the virtual ubiquitous adoption of such diagnostic practices, they are not without their limitations and accordingly, there is now increasing awareness of the importance of assessing sperm quality in order to more accurately predict a male’s fertility status. This realization raises the important question of which characteristics signify a high-quality, fertilization competent sperm cell. In this review, we reflect on recent advances in our mechanistic understanding of sperm biology and function, which are contributing to a growing armory of innovative approaches to diagnose and treat male infertility. In particular we review progress toward the implementation of precision medicine; the robust clinical adoption of which in the setting of fertility, currently lags well behind that of other fields of medicine. Despite this, research shows that the application of advanced technology platforms such as whole exome sequencing and proteomic analyses hold considerable promise in optimizing outcomes for the management of male infertility by uncovering and expanding our inventory of candidate infertility biomarkers, as well as those associated with recurrent pregnancy loss. Similarly, the development of advanced imaging technologies in tandem with machine learning artificial intelligence are poised to disrupt the fertility care paradigm by advancing our understanding of the molecular and biological causes of infertility to provide novel avenues for future diagnostics and treatments.

The domain of reproductive biology underpins our understanding of human fertility and forms an important part of the debate on the safety of wireless communication (WC) electromagnetic fields (EMFs). While studies on the effects of anthropogenic EMFs on reproduction are of clear importance, recent evidence suggests that such studies are well placed to provide much-anticipated mechanistic insights on the health impacts of EMFs. Resolution of the biophysical mechanism(s) of action is one of the most important keys required to unlock scientific progression and enable accurate assessment of health risk. Growing recourse to assisted reproductive technologies (ART) across developed nations has justifiably given rise to concern about our decreasing collective fertility as a species. While this issue is certainly multi-factorial, the rise of anthropogenic EMF exposures and especially those of WC technology has aligned with a simultaneous global decline in male semen quality parameters. This well recognized link to reproductive health clearly underlines the unique sensitivity of our reproductive systems to environmental change and has prompted investigation of the impact of novel environmental insults such as WC EMFs. The current picture of how WC EMFs impact reproduction is not yet completely clear, but the field offers strong evidence of negative impacts on the cells, tissues, and processes that influence fertility. Accordingly, here we summarize the highest quality evidence outlining effects of WC EMFs on reproductive tissues and germ cells, and based on this, we propose a plausible mechanism for the molecular nature of the interaction of WC EMF with our biology. We also highlight some of the controversies in this field, including those pertaining to policy. Against this background, we contend that, in parallel with our advancing research, revising the safety limits of anthropogenic EMF exposures to our population is warranted.

This article reports the proteomic legacy of in vivo exposure to the xenobiotic, acrylamide, on the epithelial cell population of the proximal segments of the mouse epididymis. Specifically, adult male mice were administered acrylamide (25 mg/kg bw/day) or vehicle control for five consecutive days before dissection of the epididymis. Epididymal epithelial cells were isolated from the proximal (caput) epididymal segment and subjected to quantitative proteomic analysis using multiplexed tandem mass tag (TMT) labeling coupled to mass spectrometry. Here, we report the data generated by this strategy, including the identification of 4405 caput epididymal epithelial cell proteins, approximately 6.8% of which displayed altered expression in response to acrylamide challenge. Our interpretation and discussion of these data features in the article "Acrylamide modulates the mouse epididymal proteome to drive alterations in the sperm small non-coding RNA profile and dysregulate embryo development".

Our previous studies have shown that p53 isoform expression is altered in breast cancer and related to prognosis. In particular, a high ∆40p53:p53α ratio is associated with worse disease-free survival. In this manuscript, the influence of altered Δ40p53 and p53α levels on the response to standard of care DNA-damaging agents used in breast cancer treatment was investigated in vitro. Our results revealed that a high Δ40p53:p53α ratio causes cells to respond differently to doxorubicin and cisplatin treatments. Δ40p53 overexpression significantly impairs the cells' sensitivity to doxorubicin through reducing apoptosis and DNA damage, whereas Δ40p53 knockdown has the opposite effect. Further, a high Δ40p53:p53α ratio inhibited the differential expression of several genes following doxorubicin and promoted DNA repair, impairing the cells' canonical response. Overall, our results suggest that the response of breast cancer cells to standard of care DNA-damaging therapies is dependent on the expression of p53 isoforms, which may contribute to outcomes in breast cancer.

Systemic heat stress is detrimental to sperm production and function, via the induction of oxidative stress and DNA damage. However, this phenomenon is yet to be thoroughly examined in a field setting relevant to commercial breeding horses. The duration of spermatogenesis makes direct observation of the heat-fertility relationship challenging, but anecdotal evidence suggests that some stallions are more susceptible to heat-stress than others. This study aimed to design a model capable of identifying heat-susceptible stallions, by utilising environmental monitoring, statistical modelling, and oxidative DNA damage assessments. Temperature and humidity loggers were installed in the stables and paddocks of 46 Thoroughbred stallions during the 2017 and 2018 breeding seasons. Dismount semen samples were collected weekly from these stallions (2017 n = 486; 2018 n = 318). Samples were diluted (2:1, extender:semen) and sperm concentration and motility were recorded (iSperm™). Samples were then snap frozen for oxidative DNA damage assessment, using a novel 8OHdG assay. This assay can inform on both total DNA damage, and the abundance of 8-hydroxy-2'-deoxyguanosine (8OHdG) DNA adducts—thereby measuring the proportion of damage caused by oxidative stress. Fertility results were also collated at the conclusion of each breeding season. A polynomial distributed lag model was used to identify stallions whose fertility was adversely affected by heat events, based on correlations between fertility rates (per-cycle conception and first cycle conception rates) and maximal ambient temperature and humidity. A subpopulation of 18 stallions were identified as being susceptible to heat stress (r≥ 0.50; p ≤ 0.05). Of these, six stallions (accounting for 13% of the total population) demonstrated consistent negative correlations between sperm DNA damage and fertility, and consistent positive correlations between DNA damage and ambient temperature and humidity (r ≥ 0.50; p ≤ 0.05). These findings indicate that there is a subpopulation of stallions that are particularly susceptible to heat-induced subfertility, with a further subset affected by heat- induced sperm DNA damage. This study presents a novel protocol that can effectively identify heat-sensitive stallions and could easily be implemented on commercial stud farms.

BACKGROUND:Pediatric diffuse midline gliomas (DMGs) are incurable childhood cancers. The imipridone ONC201 has shown early clinical efficacy in a subset of DMGs. However, the anticancer mechanisms of ONC201 and its derivative ONC206 have not been fully described in DMGs. METHODS:DMG models including primary human in vitro (n = 18) and in vivo (murine and zebrafish) models, and patient (n = 20) frozen and FFPE specimens were used. Drug-target engagement was evaluated using in silico ChemPLP and in vitro thermal shift assay. Drug toxicity and neurotoxicity were assessed in zebrafish models. Seahorse XF Cell Mito Stress Test, MitoSOX and TMRM assays, and electron microscopy imaging were used to assess metabolic signatures. Cell lineage differentiation and drug-altered pathways were defined using bulk and single-cell RNA-seq. RESULTS:ONC201 and ONC206 reduce viability of DMG cells in nM concentrations and extend survival of DMG PDX models (ONC201: 117 days, P = .01; ONC206: 113 days, P = .001). ONC206 is 10X more potent than ONC201 in vitro and combination treatment was the most efficacious at prolonging survival in vivo (125 days, P = .02). Thermal shift assay confirmed that both drugs bind to ClpP, with ONC206 exhibiting a higher binding affinity as assessed by in silico ChemPLP. ClpP activation by both drugs results in impaired tumor cell metabolism, mitochondrial damage, ROS production, activation of integrative stress response (ISR), and apoptosis in vitro and in vivo. Strikingly, imipridone treatment triggered a lineage shift from a proliferative, oligodendrocyte precursor-like state to a mature, astrocyte-like state. CONCLUSION:Targeting mitochondrial metabolism and ISR activation effectively impairs DMG tumorigenicity. These results supported the initiation of two pediatric clinical trials (NCT05009992, NCT04732065).

Per-fluoroalkyl and polyfluoroalkyl substances (PFAS) are a diverse group of synthetic fluorinated chemicals used widely in industry and consumer products. Due to their extensive use and chemical stability, PFAS are ubiquitous environmental contaminants and as such, form an emerging risk factor for male reproductive health. The long half-lives of PFAS is of particular concern as the propensity to accumulate in biological systems prolong the time taken for excretion, taking years in many cases. Accordingly, there is mounting evidence supporting a negative association between PFAS exposure and an array of human health conditions. However, inconsistencies among epidemiological and experimental findings have hindered the ability to definitively link negative reproductive outcomes to specific PFAS exposure. This situation highlights the requirement for further investigation and the identification of reliable biological models that can inform health risks, allowing sensitive assessment of the spectrum of effects of PFAS exposure on humans. Here, we review the literature on the biological effects of PFAS exposure, with a specific focus on male reproduction, owing to its utility as a sentinel marker of general health. Indeed, male infertility has increasingly been shown to serve as an early indicator of a range of co-morbidities such as coronary, inflammatory, and metabolic diseases. It follows that adverse associations have been established between PFAS exposure and the incidence of testicular dysfunction, including pathologies such as testicular cancer and a reduction in semen quality. We also give consideration to the mechanisms that render the male reproductive tract vulnerable to PFAS mediated damage, and discuss novel remediation strategies to mitigate the negative impact of PFAS contamination and/or to ameliorate the PFAS load of exposed individuals.