Ocean acidification (OA) together with other anthropogenic perturbations is projected to dramatically alter marine environments over the coming centuries. The vast majority of marine species reproduce by freely spawning sperm directly into the water column, where fertilisation can then either be external or a female can draw sperm into a burrow, brooding chamber or onto her external surface. Hence, sperm are now being released into rapidly changing seawater conditions. In this thesis, I firstly assess what is currently known on the potential for OA and other anthropogenic stressors to influence freely spawned sperm in marine invertebrate taxa. I then present a series of experimental chapters investigating the influence of OA, as a single stressor or in conjunction with a second stressor, copper, on sperm function, physiology and competitive fertilisation performance in a range of invertebrate taxa. My research demonstrates that sperm are vulnerable to the projected changes in seawater carbonate chemistry under OA, with responses observed at all biological levels from sperm physiology, swimming performance, fertilisation ecology and sperm competitiveness. In a multi-stressor experiment on polychaete gametes and larvae, I provide empirical evidence that changes to seawater pH under OA can alter the susceptibility of early life stages including sperm, to the common coastal pollutant copper. Sperm DNA damage increased by 150 % and larval survivorship was reduced by 44 % in combined exposures, than when exposed to copper alone. As a single stressor OA also acted to significantly reduce Arenicola marina sperm swimming speeds and fertilisation success. This work was followed up with a mechanistic investigation of A. marina sperm swimming performance under OA conditions. I found that the length of time between spawning and fertilisation can strongly influence the impact of OA on sperm performance. Key fitness-related aspects of sperm functioning declined after several hours under OA conditions, and these declines could not be explained by changes in sperm ATP content, oxygen consumption or viability. In a final set of experiments, I ran a set of paired competitive fertilisation trials in the sea urchin, Paracentrotus lividus. In addition to reducing fundamental sperm performance parameters, OA conditions affected competitive interactions between males during fertilisation, with potential implications for the proportion of offspring contributed by each male under the new conditions. This work suggests that the ‘best’ males currently may not be the most competitive under OA. Overall this body of work reveals a series of significant changes to sperm performance under OA that might act to perturb sperm functioning in future oceans.

L'obésité est une condition médicale résultant d'une accumulation excessive de dépôts adipeux. Le remodelage pathologique du tissu adipeux chez les sujets obèses pourrait conduire à l'élaboration de plusieurs problèmes de santé. Malheureusement, la prévalence de l'obésité augmente dans le monde entier et en particulier chez les jeunes hommes en âge de procréation. En outre, plusieurs études ont suggéré que les informations de l'environnement paternel comme l'obésité acquise restent dans l’épigénome du spermatozoïde et peuvent moduler le phénotype de la descendance. Pour toutes ces raisons, une compréhension plus approfondie des effets de l’obésité sur la composition moléculaire des spermatozoïdes est nécessaire. Le premier objectif de cette thèse était d'évaluer l'effet de l'obésité sur la composition moléculaire et sur la physiologie des spermatozoïdes mobiles. Les échantillons de sperme ont été obtenus à partir de 96 hommes s’adressant au centre de fertilité ‘A-clinic’, Liban. Les patients ont été classés en trois groupes : poids normal, le surpoids, et obèses. Nos résultats ont montré qu’il y a une rétention des histones plus élevée, et un ADN spermatique hypométhylé et hypohydroxymethylé, dans les spermatozoïdes mobiles des hommes obèses par rapport à ceux des hommes non-obeses. Par conséquent, les embryons issus de spermatozoïdes mobiles d'un homme obèse avaient une cinétique de division embryonnaire altérer par rapport à ceux provenant des spermatozoïdes d’un homme de poids normal / Obesity is a medical condition resulting from an excessive accumulation of adipose deposits. The pathological remodelling of the adipose tissue in obese subjects may lead to the development of several health problems. Unfortunately, the prevalence of obesity is increasing worldwide and of particular interest among young men of reproductive age. Furthermore, accumulated evidence suggests that information from paternal environment such as acquired obesity remains in the sperm epigenome and can modulate the phenotype of the offspring. Therefore, a deeper comprehension of the drawbacks of male excessive fatness on the sperm molecular composition is needed. The first aim of this thesis was to assess the impact of obesity on the molecular composition and on the physiology of the motile sperm. The semen samples were obtained from 96 men attending the A-clinic fertility center, Lebanon. Patients were categorized into three groups: normal weight, overweight, and obese. We showed that the motile sperm of obese men had abnormal levels of paternally inherited histones and hypomethylated/hypohydroxymethylated DNA as compared to normal weight men. Subsequently, the embryos derived from the motile sperm of an obese father had an altered morphokinetic patterns when compared to those derived from normal weight one. The second aim of this thesis was to evaluate the adaptive and evolutionary potential of non-genetic heritable mechanisms in experimentally controlled animal models. Using a high fat diet (HFD)-induced obesity mouse model, we have examined how feeding male mice with a high fat diet for multiple generations impacts the phenotype of the resulting mice

Assisted reproductive technology (ART) uses advanced techniques such as in vitro fertilisation (IVF) and intracytoplasmic sperm injection (ICSI) to combat human infertility. However, the success rate of ART is poor and can, at least in part, be attributed to detrimental (iatrogenic) damage incurred by gametes and embryos during laboratory treatment or manipulation, thus compromising their functional role and reducing the chances of fertilisation. The sperm plays two fundamental roles upon gamete fusion: (1) to deliver paternal genomic DNA of optimal integrity into the oocyte, and (2) to activate the oocyte to initiate embryogenesis. Protamine and phospholipase C zeta (PLCζ) are two critical sperm proteins fundamentally responsible for facilitating these two key roles, respectively. The essential role of these sperm proteins with regards to male fertility, and fertilisation outcome following ART treatment, has been widely reported. This thesis was predominantly designed to investigate the potential effects of cryopreservation and sperm immobilisation via polyvinylpyrrolidone (PVP) upon the abundance and localisation of protamine and PLCζ in mouse and human sperm, respectively. Deficiency of these proteins could lead to reduced sperm DNA integrity and oocyte activation ability, respectively. An immunofluorescent quantitative assay was first designed and optimised for the determination of protamine 1 (P1) and 2 (P2) levels in sperm. This assay demonstrated that the total levels of P1 and P2, but not the P1:P2 ratio, were significantly reduced (by approximately 50%) in mouse sperm following cryopreservation. This novel assay may represent a useful clinical tool to predict DNA integrity and help select sperm with the best quality DNA. Clinical screening of PLCζ was also carried out in the largest dataset reported to date and confirmed that total levels of PLCζ in human sperm varied significantly between samples (P ≤ 0.05). Cluster analysis led to the development of a PLCζ scoring system with significant potential as a clinical prognostic and diagnostic assay. Regression models also correlated fertilisation rate and PLCζ content in a total of 30 clinical samples. Collectively, these novel tools show significant promise as predictors of oocyte activation ability. Specific case studies involving vasectomy, oocyte activation deficiency (OAD), and globozoospermia, were identified and shown to be associated with significantly reduced levels of PLCζ (P ≤ 0.05). In two of these case studies, a single nucleotide polymorphism (SNP) was identified in the PLCζ promoter region, potentially indicating a novel mechanism for PLCζ expression in human sperm. Another case of OAD suggested the apparent deficiency of a crucial interacting factor in the oocyte, emphasising that OAD is not exclusively linked to sperm abnormalities. For the first time, efforts were made to assess whether PLCζ expression was linked to male age; total levels and the proportion of sperm exhibiting PLCζ were found not to differ significantly amongst a total of 46 males. Furthermore, in a pilot experiment, levels of PLCζ were significantly reduced (by 23% to 89%) in PVP-treated sperm from 9 controls and 3 infertile patients, with patient sperm showing higher susceptibility to the effects of PVP compared to controls. However, a more robust experiment featuring sperm from 16 fertile donors, failed to show any significant effect of PVP upon PLCζ. Collectively, data arising from this thesis generated a series of potential clinical tools to quantify protamine and PLCζ in sperm, provides strong evidence that levels of protamine are significantly reduced by cryopreservation, and has provided at least some evidence that PVP may cause detrimental effect upon the level of PLCζ in human sperm. Further work on the effects of vasectomy and the relative functional importance of the SNP detected in the PLCζ promoter are highly warranted. Further investigation and clinical translation of these findings may help to improve the success rate of ART.

Sexual reproduction entails complex co-evolution between the sexes, necessary for successful fertilization, ensuring individual and population-level fitness. Interfertility is the main criterion for species definition and understanding speciation requires detailed studies of reproductive barriers. However, many studies on reproductive barriers are constrained to infer evolutionary processes from patterns. In this thesis, I focus on a hybrid zone between collared and pied flycatchers (Ficedula albicollis and hypoleuca) on the island of Öland, and a trait that is essential for fertilization: sperm. Long-term monitoring of these species, combined with recent advances in molecular tools, allow me to study how complex on-going intersexual and interspecific interactions influence reproductive isolation in this young hybrid zone. I start by exploring the links between pre- and postmating sexual selection within collared flycatchers (paper I and II). I show that secondary sexual characters and indirect mate-choice benefits are tightly linked to physiology (paper I), and that a male’s attractiveness and dominance status dictate which sperm traits are optimal, as a male’s fertilization success depends on an interaction between sperm and display traits (paper II). I then report a source of strong postzygotic isolation between recently diverged collared and pied flycatchers: impaired spermatogenesis resulting in absence of mature sperm cells in hybrid males (paper III). I show however that pied flycatcher females, who are most exposed to hybridization, can mitigate these costs through mechanisms of cryptic female choice impairing heterospecific sperm performance, allowing them to bias paternity towards pure-species offspring (paper IV). Finally, by exploring the testes transcriptomes and sperm proteomes of both species, I highlight the importance of gene and protein regulation mechanisms in facilitating phenotypic divergence between these species (paper V). Thus, my thesis reveals complex interactions between primary and secondary sexual characters in a wild bird and suggests that mechanisms of sexual selection are tightly linked to essential physiological functions. I also show that genetic incompatibilities can evolve rapidly despite low genome-wide levels of divergence but that divergence in regulatory regions and proteins potentially allows fast evolution of molecular mechanisms impairing or preventing costly heterospecific fertilization.