School of BioSciences - Theses
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ItemPreimplantation embryo metabolism as a biomarker of embryonic viability and health( 2020)Worldwide, 12% of couples suffer from infertility and therefore rely on assisted reproductive technologies to conceive. The current success rate of an initial IVF cycle is ~20%, leaving ~80% of couples unsuccessful after their first cycle. Although success rates increase with subsequent cycles, represented by an increase in cumulative pregnancy rates, IVF treatment is expensive and can have a significant impact on the psychological wellbeing of couples. It is therefore imperative that research is focused on increasing the success rate of initial IVF cycles to reduce the time to pregnancy. The success of an IVF cycle significantly relies on the ability to select the most competent embryo from a patient’s cohort that has the greatest chance of establishing a viable pregnancy. Current embryo selection methods focus on the morphological and/or the morphokinetic development of the preimplantation embryo and preimplantation genetic testing can be utilized to ensure the embryo transferred is genetically normal. However, despite an embryo being regarded as high quality based on these selection methods, its success post-transfer is not guaranteed. Blastocyst metabolism is a key regulator of embryo development and through metaboloepigenetic interactions, embryonic health, and its assessment represents an additional biomarker that may improve the accuracy of embryo selection. A comparison between blastocyst metabolism, morphology, time-lapse annotations, artificial intelligence, chromosomal status and transfer success was conducted. High glucose uptake and high amino acid consumption were found to be associated with human blastocysts of high viability according to current methods of selection. Further, glucose uptake was significantly higher in human blastocysts that established a viable pregnancy. Genetically abnormal, or aneuploid, human embryos developed slower and were assigned lower viability scores. Additionally, blastocyst amino acid utilization appeared to be perturbed due to aneuploid associated stress. An analysis of vitrified mouse and human blastocyst pyruvate and/or glucose uptake post-warm was unable to provide a measure of viability. However, a morphological assessment of human blastocyst reexpansion post-warm revealed blastocysts with a greater degree of re-expansion were associated with higher live birth rates. Finally, using a mouse model to demonstrate how changes in embryo culture media can impact blastocyst metabolism and health, the addition of antioxidants (acetyl-L-carnitine, N-acetyl-L-cysteine and alpha-lipoic acid) to embryo culture media, individually and in combination, was investigated. A reduction in oxidative stress, regulation of blastocyst carbohydrate metabolism, lower NADH levels and improved blastocyst development were identified. Together the data presented in this thesis provides a comprehensive analysis of human blastocyst physiology and lay the foundation for the development of an algorithm incorporating morphological, morphokinetic and metabolic biomarkers which could assist in the identification of the most viable and healthy blastocyst for transfer. Such an algorithm may also be used to validate future advances in embryo culture conditions. Therefore, these data provide an opportunity to significantly improve human IVF success rates and reduce the time to pregnancy.
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ItemThe effects of artificial light at night on the behaviour and physiology of Drosophila melanogaster( 2019)Artificial light at night is one of the most pervasive and least recognised anthropogenic pollutants. Its extent and intensity is expanding globally, at an estimated rate of 2.2% per annum, such that many urban and peri-urban animals no longer experience natural darkness. The majority of animal species have evolved under bright days and comparatively dark nights, with their physiology and behaviour synchronised to this rhythm. Accordingly, disruption to this cycle is linked to a suite of behavioural and physiological shifts across taxa. Experimental and correlational evidence documenting phenotypic responses to the presence of artificial light at night are accumulating but the underlying mechanisms driving the observed negative impacts associated with artificial light at night are not resolved. A likely reason is that light at night disrupts circadian rhythms and contributes to perturbed oxidative status through its interaction with the indolamine, melatonin, a key driver of circadian rhythm and powerful antioxidant. A major and currently untested gap in the literature is whether light at night can act as a driver of evolutionary change. In this thesis, I investigated the short-term phenotypic impacts of ecologically relevant levels of artificial light at night on life history traits (throughout Chapters 2 to 5) and oxidative status (Chapters 2 and 4), using the model organism Drosophila melanogaster. I then used experimental evolution to explore whether artificial light at night can induce local adaptation in populations evolved over 15 to 25 generations (Chapter 4). Finally, using a melatonin dietary supplementation experiment, I attempted to investigate the relationship between artificial light at night and the melatonin pathway, thus hoping to demonstrate disruption to the melatonin pathway as a causal mechanism underpinning the observed phenotypic responses (Chapter 5). My results demonstrated experimentally, that individuals exposed to artificial light at night have short-term phenotypic changes, with disrupted mating behaviour, reduced fecundity and size, altered development patterns and reduced longevity in D. melanogaster (Chapters 2 to 5). Additionally, I present novel (and somewhat counter intuitive) evidence, that artificial light at night is associated with lower levels of reactive oxygen species in ovaries (Chapter 3) and, aligned with this, reduced oxidative DNA damage in female flies (Chapter 4). In contrast, I found no evidence for such effects in males (Chapters 3 and 4). Despite potential for evolutionary change, I found little evidence for adaptation in most fitness traits after evolution under artificial light at night (Chapter 4), with mating propensity the only life history trait to exhibit limited local adaptation. This suggests that the selective pressure of artificial light at night in the laboratory, may be weaker than anticipated. However, the fact that some level of adaptation was evidenced in relatively benign conditions, suggests that, in a more natural competitive environment, the selection pressure of light at night may be stronger. I was unable to conclude whether melatonin is a potential mechanism driving phenotypic variation following exposure to light at night (Chapter 5), as the melatonin doses and design used, despite replicating a previous study, resulted in negative fitness consequences (reduced longevity under dark nights and disrupted eclosion regardless of light treatment), suggesting the melatonin supplementation was potentially toxic. Nonetheless, these data potentially support the importance of circadian disruption under artificial light at night as a driver of the observed negative effects. I propose that further research is warranted to test different melatonin concentrations and dosage duration at different life stages and at different times relative to circadian rhythm. This variation in dosage regimens may be able to better define its role (and the relevant contributions of circadian disruption and oxidative status) in the effects of artificial light at night. This thesis demonstrates the negative impacts of artificial light at night in the model species, D. melanogaster and adds to the growing body of literature documenting taxa-wide damaging effects of artificial light at night on animal behaviour and physiology. As we continue to light our nights and reduce natural darkness globally, understanding the consequences and mechanisms behind the effects of artificial light at night is paramount to informed decision making around urban lighting strategies. Moreover, understanding the role of artificial light at night in driving evolutionary change, is of the utmost importance in maintaining wildlife stability in an increasingly urbanised world.
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ItemInvestigating the loci that contribute to convergent craniofacial evolution between the thylacine and canids( 2018)One of the most fundamental questions in evolutionary developmental biology is how phenotypic adaptations are controlled at the molecular level. One way we can address this question is by looking at examples of convergent evolution between distantly related species. Here we can ask the question; are similarities in morphology reflected by similarities in the genome? One of the most striking cases of convergent evolution in mammals is seen between the marsupial thylacine (or Tasmanian tiger) and placental canids (wolves, dingos and foxes) particularly in their cranial morphology. However, the extent of their morphological convergence has never been directly quantified. In my thesis I use a combination of morphological and molecular data to investigate candidate loci that may contribute to convergent craniofacial evolution between the thylacine and the canids. Using a geometric morphometric comparison of cranial shape between extinct and extant marsupial and placental mammals, I showed that the adult thylacine and canids represent a remarkable case of craniofacial convergence. By additionally CT scanning and landmarking all known thylacine pouch young specimens, I was able to demonstrate that the marsupial thylacine overcame its conserved neonatal constraints towards the end of its developmental period in the pouch. The strong similarities between the thylacine and canids are likely driven by underlying changes in cranial neural crest cells (NCCs), which are directly responsible for patterning the facial skeleton. I next investigated candidate loci that might be underpinning this extraordinary phenotypic convergence. RUNX2 is expressed in NCCs and is strongly implicated in driving facial length evolution in placental mammals. I hypothesized that similarities in the RUNX2 gene might partially explain similarities in facial shape between the thylacine and canids. However, unexpectedly, we found that the marsupials possess an invariant RUNX2 which cannot explain the diversity of facial shapes seen within marsupials nor craniofacial convergence. Instead, changes in facial length might be mediated through regulatory changes to RUNX2 expression. Using a genome-wide approach, we investigated homoplasy in protein coding genes. While overall homoplasy was extremely rare, we identified multiple thylacine/canid homoplasious amino acid substitutions in the osteogenic chromatin remodeller, CHD9, a known upstream regulator of RUNX2. We found that the amino acid substitution in the DNA binding domain resulted in differential expression and activation of RUNX2 in vitro and may act as a contributor to RUNX2-mediated craniofacial convergence. While I found evidence for changes in protein coding genes potentially contributing to convergence, the pleiotropic consequences of mutations in key developmental genes are thought to limit their evolvability. As such, we also used a genome wide approach to investigate accelerated evolution and convergence in the non-coding portion of the genome. We identified multiple putative cis-regulatory elements (CREs), including an enhancer upstream of the craniofacial TGF-β signalling receptor ACVR2A, also critical in NCCs. We found that the thylacine enhancer was able to drive craniofacial expression in the mouse and is a potential candidate mediating convergent craniofacial evolution. This finding suggests CREs may also play important roles in adaptive evolution and convergence. In this thesis I find support for protein coding and CRE evolution driving convergent craniofacial similarities. This supports my hypothesis that convergence targets genes and CREs in the NCCs directing craniofacial convergence between the thylacine and canids.
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ItemThe physiological effects of artificial light at night on the Australian black field cricket( 2018)The presence of artificial light at night (ALAN) is one of the fastest growing, most pervasive and, until recently, under-appreciated forms of global pollution. Current ALAN levels in urban environments are associated with changes to animal behaviour, dramatic shifts in the timing of life history events, reductions in individual fitness and disrupted physiological processes, including immune function. This thesis explores the physiological effects of ecologically relevant levels of ALAN on a model invertebrate species, the Australian black field cricket, Teleogryllus commodus. In Chapter 1, I reviewed the literature with a particular emphasis on the physiological effects of ALAN, including growth, survival, reproductive success, and immune function. I also speculate as to the potential mechanistic links behind these ALAN induced biological effects. In Chapter 2, I explored experimentally the effects of ecologically relevant levels of ALAN (1, 10 and 100 lux) on life history and fitness traits of the black field cricket. Under controlled laboratory conditions, I reared crickets from egg to adult in an environment with either no ALAN (0 lux) or one of the above dim-ALAN intensities and assessed the consequences of ALAN for growth, survival and reproductive success. I demonstrated that egg hatch, adult survival and reproductive measures were largely unaffected by the presence of ALAN, however juvenile development time was longer and adults were larger when crickets were exposed to any light at night (1, 10 or 100 lux). In Chapter 3, I examined the effects of ALAN (1, 10 and 100 lux) on three key measures of adult immune function (haemocyte concentration, lytic activity, and phenoloxidase activity). The presence of any ALAN (1, 10 or 100 lux) had a clear negative effect on the cellular immune response. Specifically, individuals exposed to any ALAN were unable to increase their haemocyte concentration in response to a stressor challenge. In Chapter 4, I investigated a novel method for the measurement of circulating melatonin in small samples of cricket haemolymph using high-performance liquid chromatography tandem mass spectrometry, with methyl tert-butyl ether (MTBE)/ethyl acetate as an extraction agent. The calibration curve for melatonin was linear in the range of 0.25 and 10 pM (R2 = 0.999), and the limit of detection was 0.25pM. When applied to a set of pilot data from crickets reared under different ALAN environments (0, 1, 10, and 100 lux), the results were however inconclusive, due to small sample sizes. In Chapter 5, I discuss the significance of these findings and their ecological implications. My thesis advances our understanding of the biological ef fects of ALAN for invertebrates, a key taxon contributing to ecological community structure and composition. It is one of the first set of studies to simultaneously investigate multiple traits in the same individuals exposed to lifelong ALAN, and to assess changes in immune function throughout their adult life. Combined, the results presented demonstrate a disruption to physiological processes, and highlight the potential for ALAN to alter the phenology of communities and reduce the overall fitness of individuals.
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ItemDefining the master regulator of urethral closure in mouse( 2017)Hypospadias is the ectopic placement of the urethral opening on the underside of the penis and is one of the most common developmental abnormalities in humans, occurring in approximately 1 in every 125 live male births. In addition, we have observed a doubling in the incidence of hypospadias over the past several decades suggesting an environmental component likely in the form of estrogen mimicking chemicals generally referred to as environmental endocrine disruptors (EEDs). Current models fail to explain these observations. The goal of this thesis is to produce a theory that describes the development and genetic regulation of urethral closure, and use it to explain the aetiology, spectrum, and rise in incidence of hypospadias observed in humans. The work presented in this thesis was performed using a novel mouse model (OVE442) with isolated hypospadias. This model was used to define the role of the urorectal septum (URS) during urethral closure. The process of urethral closure is generally thought to occur by tissue fusion. However, we provide immunohistological evidence that suggests the urethra is internalized by growth of the URS, which contributes tissue to the ventral aspect of the penis during embryonic development. The OVE442 model was next used to define a key regulator of the URS during urethral closure. Initial characterization of a genomic mutation in OVE442 model led us to discover a long non-coding RNA, designated Leat1, which was deleted near EfnB2. Loss of signalling through the EPHRINB2 protein was previously shown to cause severe hypospadias in mouse, however little is known about EfnB2 gene regulation during urethral closure. Leat1 was characterized, functionally examined, and shown to regulate EfnB2 expression through direct interaction with the EPHRINB2 protein. We further showed that Leat1 expression is differentially regulated in males and females, and that it is supressed by estrogen. These results showed that EfnB2 drives growth of the URS during urethral closure and provided the first experimental evidence revealing the genetic mechanism that causes male and female urethral anatomy to diverge. These observations were used together with our anatomical descriptions to produce a developmental theory that explains urethral formation in mouse. We extended our understanding further by using comparative time series RNA-Seq to describe global transcription and ChIP-Seq to identify genes actively regulated by estrogen and androgen during urethral. From these data, we identify potential urethral closure genes downstream of Leat1 and EfnB2 including genes that are likely responsive to sex hormones. This work has provided fundamental insights on the anatomy and genetic regulation of urethral closure. I have shown that male urethral closure is driven by growth of the URS and that this growth is regulated by the long non-coding RNA Leat1 in mouse. Furthermore, I have produced a list of potential EED targets that may lead to better understanding the causes of hypospadias. Through this work I have produced a theory that explains the spectrum of urethral malformations observed in human, defects associated with hypospadias such as chordee, and the genetic mechanism that is likely disrupted by EEDs. These findings fundamentally change the way we consider urethral development and may help to find ways to reduce the incidence or prevent hypospadias in humans.
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ItemMale obesity negatively affects fecundity; reducing seminal plasma quality, embryo physiology and pregnancy health( 2015)Australia is echoing trends seen across the globe with steadily increasing rates of obesity. Worldwide, the rate of obesity has nearly doubled in the past three decades, and in particular, obesity amongst men of reproductive age has tripled. While an association between excessive weight and subfertility is now largely recognised, the consequences of male obesity on reproductive function are poorly understood. The aim of the work carried out in this thesis was to investigate the effects of male obesity on preimplantation embryo development and subsequent fetal health, and identify changes in the spermatozoa and seminal plasma of obese men that might result in compromised pregnancies. The glut of data regarding the effect of male obesity on fertility, implantation and pregnancy health originates from fertility clinics, with study cohorts biased towards a subfertile population and influenced by numerous lifestyle factors. Comparatively, animal models are free of confounding variables. Male C57Bl/6 mice were fed a high fat ‘Western fast food’ style diet to induce obesity, and used for sample generation. Obese mice gained significantly more weight than their normal counterparts and had significantly increased body fat. Appropriately timed development is a key indicator of embryo quality. Embryos were examined under time-lapse microscopy to assess timing of cleavage events. Embryos derived from obese males were slower than normal from as early as pronuclear fusion. These embryos also had aberrant preimplantation metabolism and mitochondrial membrane potential, as well as disproportionate allocation of cells to the inner cell mass and trophectoderm. When these embryos were transferred to recipient female mice, embryos derived from obese fathers resulted in a successful pregnancy less often than their normal counterparts. Further examination revealed this to be the result of poor embryo implantation. Modelling attachment and the initial stages of implantation in vitro showed embryos derived from obese fathers attached to the extracellular matrix at the same rate as normal, but had decreased outgrowth over the culture period. When embryos derived from obese fathers did result in a successful pregnancy, placentas were significantly smaller than normal and fetal growth was restricted. While male and female fetuses appeared equally affected morphologically, molecular changes as a result of paternal obesity were identified as sex-specific. Female placentas derived from obese fathers had significantly increased global DNA methylation, and male placentas had altered RNA levels of a number of key metabolic and apoptotic genes. These changes in embryo, fetal and placental development do not appear to be the result of gross morphological changes to the spermatozoa of obese males. Total sperm count, motility and acrosomal reaction were all unaffected by obesity. However, RNA levels of cytochrome c oxidase subunit IV isoform 1 were significantly higher in the spermatozoa of obese males compared to normal. Furthermore, the protein and carbohydrate milieu of the seminal plasma was significantly affected by obesity. The findings within this thesis demonstrate the significant effect of male obesity on reproductive fitness, affecting preimplantation embryo development and subsequent pregnancy health, as well as seminal plasma composition. Furthermore, this work highlights the importance of paternal health prior to conception, a factor often overlooked in the making of a healthy baby.