Associate Professor Geoffry De Iuliis

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 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 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 cyto-toxic 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 lipoxygen-ase-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 arachi-donic 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.

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.

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.

Reactive oxygen species (ROS) are implicated in all aspects of cellular functions. While the importance of ROS as signalling molecules is well described, ROS are also associated with stress pathologies. Within the reproduction field, there are associations with reduced fertility as a result of lipid peroxidation, protein dysfunction, premature cell death and DNA damage which readily occur in spermatozoa. These oxidative insults can arise in vivo, or in vitro as a result of sperm storage, purification and processing. Following a brief description of the production, homeostasis and functions of ROS in mammalian sperm function, this review paper will focus on describing the predominant sources of ROS in the ejaculate, the effects of ROS on a cellular and molecular level, and the actions of ROS from the whole animal perspective. There is highlighting of some studies, which have revealed the mechanisms for these observations, along with some strategies to ameliorate or prevent the instigation of the oxidative stress cascade before irreversible damage to spermatozoa occurs.

STUDY QUESTION: What are the mechanisms by which the preparation of spermatozoa on discontinuous density gradients leads to an increase in oxidative DNA damage? SUMMARY ANSWER: The colloidal silicon solutions that are commonly used to prepare human spermatozoa for assisted reproduction technology (ART) purposes contain metals in concentrations that promote free radical-mediated DNA damage. WHAT IS KNOWN ALREADY: Sporadic reports have already appeared indicating that the use of colloidal silicon-based discontinuous density gradients for sperm preparation is occasionally associated with the induction of oxidative DNA damage. The cause of this damage is however unknown. STUDY DESIGN, SIZE, DURATION: This study comprised a series of experiments designed to: (i) confirm the induction of oxidativeDNA damage in spermatozoa prepared on commercially available colloidal silicon gradients, (ii) compare the levels of damage observed with alterative sperm preparation techniques including an electrophoretic approach and (iii) determine the cause of the oxidative DNA damage and develop strategies for its prevention. The semen samples employed for this analysis involved a cohort of >50 unselected donors and at least three independent samples were used for each component of the analysis. PARTICIPANTS/MATERIALS, SETTING, METHODS: The setting was a University biomedical science laboratory. The major techniques employed were: (i) flow cytometry to study reactive oxygen species generation, lipid peroxidation and DNA damage, (ii) computeraided sperm analysis to measure spermmovement and (iii) inductively coupled mass spectrometry to determine the elemental composition of sperm preparation media. MAIN RESULTS AND THE ROLE OF CHANCE: Oxidative DNA damage is induced in spermatozoa prepared on PureSperm® discontinuous colloidal silicon gradients (P < 0.001 versus repeated centrifugation) because this medium contains metals, particularly Fe, Al and Cu, which are known to promote free radical generation in the immediate vicinity of DNA. This damage can be significantly accentuated by reducing agents, such as ascorbate (P < 0.001) and inhibited by selective chelation (P < 0.001). This problem is not confined to PureSperm®; analysis of additional commercial sperm preparation media revealed that metal contamination is a relatively constant feature of such products. LIMITATIONS, REASONS FOR CAUTION: While the presence of metals, particularly transition metals, may exacerbate the levels of oxidative DNA damage seen in human spermatozoa, the significance of such damage has not yet been tested in suitably powered clinical trials. WIDER IMPLICATIONS OF THE FINDINGS: The results explain why the preparation of spermatozoa on discontinuous colloidal silicon gradients can result in oxidative DNA damage. The results are of immediate relevance to the development of safe, effective protocols for the preparation of spermatozoa for ART purposes. STUDY FUNDING/COMPETING INTEREST(S): The studywas funded by the Australian Health and Medical ResearchCouncil. One of the authors (R.J.A.) has had a consultantship with a biotechnology company, NuSep, interested in the development of electrophoretic methods of sperm preparation. He has no current financial interest in this area. None of the other authors have a conflict of interest to declare.

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 levels 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 conceivedin vitro and serves as a driver for current research into the origins of free radical generation in the germ line. © 2014 AJA, SIMM & SJTU. All rights reserved.