Exploring facets of sperm function and their potential as conduits of systemic ill health in men

<p dir="ltr">Infertility is defined as the inability to conceive after 12 months of unprotected, regular intercourse. Infertility affects as many as 15-20% of couples globally, and male infertility accounts for up to half of all instances. For over a decade the body of evidence has grown linking male infertility to both prevalent and incident diagnoses of chronic illnesses. This trend is observed for chronic illnesses in the categories of oncological disease, cardiovascular disease, metabolic disorders and autoimmune diseases in men who have previously received a subfertility diagnosis. This trend of an increased rate of concomitant chronic illnesses (comorbidities) has been demonstrated repeatedly between infertile men and fertile comparators in retrospective and cohort analyses for numerous stratification criteria, including childlessness as a proxy for infertility, decreasing sperm count, decreasing sperm motility, and pathologies including azoospermia and testicular hypofunction. Despite this, no clear mechanism underpinning a common aetiology between male infertility and its comorbidities has been established. Moreover, our understanding of the underlying molecular nature of male infertility itself, especially in cases of idiopathic infertility, is still lacking. Given these important knowledge gaps, the overall aims of the studies described within this thesis were to increase our molecular understanding of infertility, explore avenues for novel treatments focusing on modulation of the lipid oxidation enzyme arachidonate 15-lipoxygenase (ALOX15) and investigate a role for lipid oxidation as a potential molecular conduit between male infertility and chronic illness. </p><p dir="ltr">In completing these studies, we produced the first putative human sperm kinome. Using in silico prediction tools paired with a high throughput phosphoproteomic method, EasyPhos, we assembled a repository of both predicted kinases and those detected through mass spectrometry to compliment previously validated kinases with known roles in sperm capacitation. To validate our putative human sperm kinome, we targeted several predicted kinase candidates through pharmacological inhibition under capacitating conditions in vitro. In doing so, we demonstrated a previously unexplored role for polo like kinase 1 (PLK1) in human sperm capacitation. Specifically, inhibition of PLK1 reduced capacitation associated increases in progressive motility, protein tyrosine phosphorylation, and suppressed the ability of sperm to undergo either a spontaneous or progesterone induced acrosome reaction.</p><p><br></p><p dir="ltr">Building on this work, we used our optimised phosphoproteomic protocol and established a kinase prediction workflow to explore the ramifications of exogenous application of 4-hydroxy-2-nonenal (4HNE), a reactive lipid aldehyde produced through lipid peroxidation, on sperm cell signalling. Here, we demonstrated for the first time in sperm that 4HNE has the capacity to dysregulate protein phosphorylation. To aid future exploration of the mechanisms controlling this sensitivity to 4HNE, we have produced a repository of kinases that may be driving the phosphorylation changes observed. Excitingly, the inhibition of ALOX15, a lipid modulating enzyme and significant contributor to the production of reactive lipid aldehydes in sperm, during 4HNE exposure conferred protection against the 4HNE associated dysregulation of protein phosphorylation signatures and prevented the lipid aldehyde modification of several proteins with key roles in sperm function, including zona pellucida binding protein 1 (ZPBP) and A-kinase anchor protein 4 (AKAP4). </p><p dir="ltr">Finally, in seeking to explore the relationship between lipid damage, male infertility, and chronic illness, we generated a constitutive global Alox15 overexpressing mouse line (Alox15tg/+) and characterised the fertility and made tissue specific observations pertaining to these animals during ageing. In addition to significantly reduced bodyweight compared to wildtype mice, Alox15tg/+ mice had decreased perigonadal fat deposition and increased liver weight, two indicators of severely disrupted metabolism. In a comprehensive assessment of fertility, aged (26-week old) Alox15tg/+ were subfertile, exhibiting decreased testis weight, decreased daily sperm production, and an overall decrease in pregnancies per pair of mice. Histological examination of the testes further indicated that Alox15tg/+ mice had an increased propensity for spermatogenic failure among both young (12-week old) and aged animals. Despite this, the spermatozoa collected from Alox15tg/+ exhibited typical motility, morphology and capacitation. In contrast to their normal appearance, deeper flow cytometric evaluation of Alox15tg/+ sperm revealed a decrease in membrane fluidity and increased reactive oxygen species production compared to sperm from wildtype animals. Thus, this study sheds light on the role of ALOX15 in both fertility and health and provides a tractable model for further investigation to explore lipid damage as a conduit between somatic and reproductive health.</p><p dir="ltr">Taken together, this body of work provides the field with new insights into sperm function, in particular the intricate roles of phosphorylation as a cellular response to oxidative stress and as a driver of capacitation. It is my hope that these findings can act as a foundation in the pursuit of much needed high resolution insight into both male infertility and its relationship with ill health.</p>