Assessment of the impact of direct in vitro PFAS treatment on mouse spermatozoa.

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.

New horizons in human sperm selection for assisted reproduction.

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.

Alterations in the p53 isoform ratio govern breast cancer cell fate in response to DNA damage.

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.

Quantitative proteomic dataset of mouse caput epididymal epithelial cells exposed to acrylamide in vivo.

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".

Imipridones affect tumor bioenergetics and promote cell lineage differentiation in diffuse midline gliomas.

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).

Pharmaco-proteogenomic profiling of pediatric diffuse midline glioma to inform future treatment strategies.

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.

Acrylamide modulates the mouse epididymal proteome to drive alterations in the sperm small non-coding RNA profile and dysregulate embryo development.

Paternal exposure to environmental stressors elicits distinct changes to the sperm sncRNA profile, modifications that have significant post-fertilization consequences. Despite this knowledge, there remains limited mechanistic understanding of how paternal exposures modify the sperm sncRNA landscape. Here, we report the acute sensitivity of the sperm sncRNA profile to the reproductive toxicant acrylamide. Furthermore, we trace the differential accumulation of acrylamide-responsive sncRNAs to coincide with sperm transit of the proximal (caput) segment of the epididymis, wherein acrylamide exposure alters the abundance of several transcription factors implicated in the expression of acrylamide-sensitive sncRNAs. We also identify extracellular vesicles secreted from the caput epithelium in relaying altered sncRNA profiles to maturing spermatozoa and dysregulated gene expression during early embryonic development following fertilization by acrylamide-exposed spermatozoa. These data provide mechanistic links to account for how environmental insults can alter the sperm epigenome and compromise the transcriptomic profile of early embryos.

Proteomic analysis of koala (phascolarctos cinereus) spermatozoa and prostatic bodies.

The aims of this study were to investigate the proteome of koala spermatozoa and that of the prostatic bodies with which they interact during ejaculation. For this purpose, spermatozoa and prostatic bodies were fractionated from the semen of four male koalas and analysed by HPLC MS/MS. This strategy identified 744 sperm and 1297 prostatic body proteins, which were subsequently attributed to 482 and 776 unique gene products, respectively. Gene ontology curation of the sperm proteome revealed an abundance of proteins mapping to the canonical sirtuin and 14-3-3 signalling pathways. By contrast, protein ubiquitination and unfolded protein response pathways dominated the equivalent analysis of proteins uniquely identified in prostatic bodies. Koala sperm proteins featured an enrichment of those mapping to the functional categories of cellular compromise/inflammatory response, whilst those of the prostatic body revealed an over-representation of molecular chaperone and stress-related proteins. Cross-species comparisons demonstrated that the koala sperm proteome displays greater conservation with that of eutherians (human; 93%) as opposed to reptile (crocodile; 39%) and avian (rooster; 27%) spermatozoa. Together, this work contributes to our overall understanding of the core sperm proteome and has identified biomarkers that may contribute to the exceptional longevity of koala spermatozoa during ex vivo storage.

A novel role for milk fat globule-EGF factor 8 protein (MFGE8) in the mediation of mouse sperm-extracellular vesicle interactions.

Spermatozoa transition to functional maturity as they are conveyed through the epididymis, a highly specialized region of the male excurrent duct system. Owing to their transcriptionally and translationally inert state, this transformation into fertilization competent cells is driven by complex mechanisms of intercellular communication with the secretory epithelium that delineates the epididymal tubule. Chief among these mechanisms are the release of extracellular vesicles (EV), which have been implicated in the exchange of varied macromolecular cargo with spermatozoa. Here, we describe the optimization of a tractable cell culture model to study the mechanistic basis of sperm-extracellular vesicle interactions. In tandem with receptor inhibition strategies, our data demonstrate the importance of milk fat globule-EGF factor 8 (MFGE8) protein in mediating the efficient exchange of macromolecular EV cargo with mouse spermatozoa; with the MFGE8 integrin-binding Arg-Gly-Asp (RGD) tripeptide motif identified as being of particular importance. Specifically, complementary strategies involving MFGE8 RGD domain ablation, competitive RGD-peptide inhibition and antibody-masking of alpha V integrin receptors, all significantly inhibited the uptake and redistribution of EV-delivered proteins into immature mouse spermatozoa. These collective data implicate the MFGE8 ligand and its cognate integrin receptor in the mediation of the EV interactions that underpin sperm maturation.

Gross and microanatomy of the male reproductive duct system of the saltwater crocodile Crocodylus porosus.

Information on the morphology and histology of the male reproductive system of the Crocodylia species is necessary to determine the role of these tissues in the production of functional spermatozoa. Accordingly, in this study we examined the gross morphology and microanatomy of the testis and the male excurrent duct system through which spermatozoa pass before ejaculation. The data demonstrate that the reproductive system in male saltwater crocodiles comprises paired testes, which convey spermatozoa distally via the rete testis into an excurrent duct system comprising ductuli efferentes, ductuli epididymides, ductus epididymidis and ductus deferens. The epithelium delineating the male tract was dominated by non-ciliated and ciliated cells structured into a simple columnar lining of the ductuli efferentes and ductuli epididymides, through to the high pseudostratified columnar epithelium of the ductus epididymidis and ductus deferens. The morphology and histochemical staining of these ducts suggest their involvement in seminal fluid production and/or its modification, which likely contributes to the nourishment, protection and/or storage of crocodile spermatozoa. As a reflection of their common Archosaurs ancestry, the overall structural characteristics we describe for the crocodile male excurrent duct system share closer similarities to those of the Aves than other clades within the Reptilia class or Mammalia.

Post-testicular sperm maturation in the saltwater crocodile Crocodylus porosus: assessing the temporal acquisition of sperm motility.

Conservation efforts to secure the long-term survival of crocodilian species would benefit from the establishment of a frozen sperm bank in concert with artificial breeding technologies to maintain genetic diversity among captive assurance populations. Working towards this goal, our research has focused on the saltwater crocodile Crocodylus porosus as a tractable model for understanding crocodilian sperm physiology. In extending our systematic characterisation of saltwater crocodile spermatozoa, in this study we examined the development of motility during sperm transport through the excurrent duct system of the male crocodile. The results show that approximately 20% of crocodile testicular spermatozoa are immediately motile but experience a gradient of increasing motility (percentage motile and rate of movement) as they transit the male reproductive tract (epididymis). Moreover, we confirmed that, as in ejaculated crocodile spermatozoa, increased intracellular cAMP levels promoted a significant and sustained enhancement of sperm motility regardless of whether the cells were isolated from the testis or epididymis. Along with the development of artificial reproductive technologies, this research paves the way for the opportunistic recovery, storage and potential utilisation of post-mortem spermatozoa from genetically valuable animals.

Preclinical and clinical evaluation of German-sourced ONC201 for the treatment of H3K27M-mutant diffuse intrinsic pontine glioma.

BACKGROUND: Diffuse intrinsic pontine glioma (DIPG) is a fatal childhood brainstem tumor for which radiation is the only treatment. Case studies report a clinical response to ONC201 for patients with H3K27M-mutant gliomas. Oncoceutics (ONC201) is only available in the United States and Japan; however, in Germany, DIPG patients can be prescribed and dispensed a locally produced compound-ONC201 German-sourced ONC201 (GsONC201). Pediatric oncologists face the dilemma of supporting the administration of GsONC201 as conjecture surrounds its authenticity. Therefore, we compared GsONC201 to original ONC201 manufactured by Oncoceutics Inc. METHODS: Authenticity of GsONC201 was determined by high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy. Biological activity was shown via assessment of on-target effects, in vitro growth, proliferation, and apoptosis analysis. Patient-derived xenograft mouse models were used to assess plasma and brain tissue pharmacokinetics, pharmacodynamics, and overall survival (OS). The clinical experience of 28 H3K27M+ mutant DIPG patients who received GsONC201 (2017-2020) was analyzed. RESULTS: GsONC201 harbored the authentic structure, however, was formulated as a free base rather than the dihydrochloride salt used in clinical trials. GsONC201 in vitro and in vivo efficacy and drug bioavailability studies showed no difference compared to Oncoceutics ONC201. Patients treated with GsONC201 (n = 28) showed a median OS of 18 months (P = .0007). GsONC201 patients who underwent reirradiation showed a median OS of 22 months compared to 12 months for GsONC201 patients who did not (P = .012). CONCLUSIONS: This study confirms the biological activity of GsONC201 and documents the OS of patients who received the drug; however, GsONC201 was never used as a monotherapy.

Glycerophospholipids protect stallion spermatozoa from oxidative damage in vitro.

Stallion sperm membranes comprise a high proportion of polyunsaturated fatty acids, making stallion spermatozoa especially vulnerable to peroxidative damage from reactive oxygen species generated as a by-product of cell metabolism. Membrane lipid replacement therapy with glycerophospholipid (GPL) mixtures has been shown to reduce oxidative damage in vitro and in vivo. The aims of this study were to test the effects of a commercial preparation of GPL, NTFactor® Lipids, on stallion spermatozoa under oxidative stress. When oxidative damage was induced by the addition of arachidonic acid to stallion spermatozoa, the subsequent addition of GPL reduced the percentage of 4-hydroxynonenal (4-HNE; a key end product of lipid peroxidation) positive cells (32.9 ± 2.7 vs 20.9 ± 2.3%; P ≤ 0.05) and increased the concentration of 4-HNE within the spent media (0.026 ± 0.003 vs 0.039 ± 0.004 µg/mL; P ≤ 0.001), suggesting that oxidized lipids had been replaced by exogenous GPL. Lipid replacement improved several motility parameters (total motility: 2.0 ± 1.0 vs 68.8 ± 2.9%; progressive motility: 0 ± 0 vs 19.3 ± 2.6%; straight line velocity: 9.5 ± 2.1 vs 50.9 ± 4.1 µm/s; curvilinear velocity: 40.8 ± 10 vs 160.7 ± 7.8 µm/s; average path velocity: 13.4 ± 2.9 vs 81.9 ± 5.9 µm/s; P ≤ 0.001), sperm viability (13.5 ± 2.9 vs 80.2 ± 1.6%; P ≤ 0.001) and reduced mitochondrial ROS generation (98.2 ± 0.6 vs 74.8 ± 6.1%; P ≤ 0.001). Supplementation with GPL during 17°C in vitro sperm storage over 72 h improved sperm viability (66.4 ± 2.6 vs 78.1 ± 2.9%; P ≤ 0.01) and total motility (53 ± 5.6 vs 66.3 ± 3.5%; P ≤ 0.05). It is concluded that incubation of stallion spermatozoa with sub-µm-sized GPL micelles results in the incorporation of exogenous GPL into sperm membranes, diminishing lipid peroxidation and improving sperm quality in vitro. LAY SUMMARY: Sperm collection and storage is an important step in many artificial insemination and in vitro fertilization regimes for several species, including humans and horses. The sperm membrane, which acts as a protective outer barrier, is made up of fatty acid-containing molecules - called phospholipids. These phospholipids may become damaged by waste products generated by the cell, such as hydrogen peroxide, during non-chilled sperm storage. We aimed to determine if sperm cells were able to repair this membrane damage by supplementing them with phospholipids during non-chilled storage. Sperm was collected from five miniature stallions by artificial vagina, and then supplemented with phospholipids during 72 h sperm storage at 17°C. Our studies show that when stallion sperm are supplemented with phospholipids in vitro, they are able to remove their damaged membrane phospholipids and swap them for undamaged ones, aiding in resistance to cellular waste and improving cell health and potential fertility.

Assessment of the Emerging Threat Posed by Perfluoroalkyl and Polyfluoroalkyl Substances to Male Reproduction in Humans.

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.

Molecular insights into the divergence and diversity of post-testicular maturation strategies.

Competition to achieve paternity has coerced the development of a multitude of male reproductive strategies. In one of the most well-studied examples, the spermatozoa of all mammalian species must undergo a series of physiological changes as they transit the male (epididymal maturation) and female (capacitation) reproductive tracts prior to realizing their potential to fertilize an ovum. However, the origin and adaptive advantage afforded by these intricate processes of post-testicular sperm maturation remain to be fully elucidated. Here, we review literature pertaining to the nature and the physiological role of epididymal maturation and subsequent capacitation in comparative vertebrate taxa including representative species from the avian, reptilian, and mammalian lineages. Such insights are discussed in terms of the framework they provide for helping to understand the evolutionary significance of post-testicular sperm maturation.

The Sins of Our Forefathers: Paternal Impacts on De Novo Mutation Rate and Development.

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.

Good Cop, Bad Cop: Defining the Roles of Δ40p53 in Cancer and Aging.

The tumour suppressor p53 is essential for maintaining DNA integrity, and plays a major role in cellular senescence and aging. Understanding the mechanisms that contribute to p53 dysfunction can uncover novel possibilities for improving cancer therapies and diagnosis, as well as cognitive decline associated with aging. In recent years, the complexity of p53 signalling has become increasingly apparent owing to the discovery of the p53 isoforms. These isoforms play important roles in regulating cell growth and turnover in response to different stressors, depending on the cellular context. In this review, we focus on Δ40p53, an N-terminally truncated p53 isoform. Δ40p53 can alter p53 target gene expression in both a positive and negative manner, modulating the biological outcome of p53 activation; it also functions independently of p53. Therefore, proper control of the Δ40p53: p53 ratio is essential for normal cell growth, aging, and responses to cancer therapy. Defining the contexts and the mechanisms by which Δ40p53 behaves as a "good cop or bad cop" is critical if we are to target this isoform therapeutically.

Mechanistic Insight into the Regulation of Lipoxygenase-Driven Lipid Peroxidation Events in Human Spermatozoa and Their Impact on Male Fertility.

A prevalent cause of sperm dysfunction in male infertility patients is the overproduction of reactive oxygen species, an attendant increase in lipid peroxidation and the production of cytotoxic reactive carbonyl species such as 4-hydroxynonenal. Our previous studies have implicated arachidonate 15-lipoxygenase (ALOX15) in the production of 4-hydroxynonenal in developing germ cells. Here, we have aimed to develop a further mechanistic understanding of the lipoxygenase-lipid peroxidation pathway in human spermatozoa. Through pharmacological inhibition studies, we identified a protective role for phospholipase enzymes in the liberation of peroxidised polyunsaturated fatty acids from the human sperm membrane. Our results also revealed that arachidonic acid, linoleic acid and docosahexanoic acid are key polyunsaturated fatty acid substrates for ALOX15. Upon examination of ALOX15 in the spermatozoa of infertile patients compared to their normozoospermic counterparts, we observed significantly elevated levels of ALOX15 protein abundance in the infertile population and an increase in 4-hydroxynonenal adducts. Collectively, these data confirm the involvement of ALOX15 in the oxidative stress cascade of human spermatozoa and support the notion that increased ALOX15 abundance in sperm cells may accentuate membrane lipid peroxidation and cellular dysfunction, ultimately contributing to male infertility.

Whole-body exposures to radiofrequency-electromagnetic energy can cause DNA damage in mouse spermatozoa via an oxidative mechanism.

Artificially generated radiofrequency-electromagnetic energy (RF-EME) is now ubiquitous in our environment owing to the utilization of mobile phone and Wi-Fi based communication devices. While several studies have revealed that RF-EME is capable of eliciting biological stress, particularly in the context of the male reproductive system, the mechanistic basis of this biophysical interaction remains largely unresolved. To extend these studies, here we exposed unrestrained male mice to RF-EME generated via a dedicated waveguide (905 MHz, 2.2 W/kg) for 12 h per day for a period of 1, 3 or 5 weeks. The testes of exposed mice exhibited no evidence of gross histological change or elevated stress, irrespective of the RF-EME exposure regimen. By contrast, 5 weeks of RF-EME exposure adversely impacted the vitality and motility profiles of mature epididymal spermatozoa. These spermatozoa also experienced increased mitochondrial generation of reactive oxygen species after 1 week of exposure, with elevated DNA oxidation and fragmentation across all exposure periods. Notwithstanding these lesions, RF-EME exposure did not impair the fertilization competence of spermatozoa nor their ability to support early embryonic development. This study supports the utility of male germ cells as sensitive tools with which to assess the biological impacts of whole-body RF-EME exposure.

Mechanisms of tethering and cargo transfer during epididymosome-sperm interactions.

BACKGROUND: The mammalian epididymis is responsible for the provision of a highly specialized environment in which spermatozoa acquire functional maturity and are subsequently stored in preparation for ejaculation. Making important contributions to both processes are epididymosomes, small extracellular vesicles released from the epididymal soma via an apocrine secretory pathway. While considerable effort has been focused on defining the cargo transferred between epididymosomes and spermatozoa, comparatively less is known about the mechanistic basis of these interactions. To investigate this phenomenon, we have utilized an in vitro co-culture system to track the transfer of biotinylated protein cargo between mouse epididymosomes and recipient spermatozoa isolated from the caput epididymis; an epididymal segment that is of critical importance for promoting sperm maturation. RESULTS: Our data indicate that epididymosome-sperm interactions are initiated via tethering of the epididymosome to receptors restricted to the post-acrosomal domain of the sperm head. Thereafter, epididymosomes mediate the transfer of protein cargo to spermatozoa via a process that is dependent on dynamin, a family of mechanoenzymes that direct intercellular vesicle trafficking. Notably, upon co-culture of sperm with epididymosomes, dynamin 1 undergoes a pronounced relocation between the peri- and post-acrosomal domains of the sperm head. This repositioning of dynamin 1 is potentially mediated via its association with membrane rafts and ideally locates the enzyme to facilitate the uptake of epididymosome-borne proteins. Accordingly, disruption of membrane raft integrity or pharmacological inhibition of dynamin both potently suppress the transfer of biotinylated epididymosome proteins to spermatozoa. CONCLUSION: Together, these data provide new mechanistic insight into epididymosome-sperm interactions with potential implications extending to the manipulation of sperm maturation for the purpose of fertility regulation.