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There are currently over 1.9 billion adults globally who are overweight or obese. As the prevalence of obesity increases worldwide this includes women of childbearing age. Over half of all women of reproductive age in the UK are currently classed as overweight or obese. Obesity during pregnancy is known to have a negative impact on maternal health, pregnancy outcome, and the long-term cardiometabolic health of her offspring. Maternal interventions in obese pregnancy are needed to prevent transmission of poor cardiometabolic outcomes between mother and child. Maternal exercise in obese pregnancy in both humans and animal models has shown success for the prevention of adverse outcomes. As maternal obesity also results in maternal, placental, and fetal oxidative stress, antioxidant interventions to the mother may be another effective treatment. Human and mouse models of maternal obesity have shown that antioxidant supplementation can prevent an adverse offspring cardiometabolic phenotype. Our lab has previously shown that prenatal exposure to maternal obesity results in long-term consequences for offspring cardiometabolic health in adult male offspring. However, more recent studies have highlighted that there can be sex-specific effects of the maternal environment on offspring health. In the Ozanne lab mouse model of maternal diet-induced obesity, the cardiac phenotype in female offspring is still to be established. There has also been limited exploration of the effects of maternal obesity on either male or female cardiac function in fetal life. Furthermore, there is a lack of literature on the effects of exercise and antioxidant interventions in obese pregnancy on offspring cardiac function. The aims of this thesis are therefore: (1) Chapter 3 - to characterise the sex-specific effects of maternal obesity on cardiac dysfunction in 8-week-old male and female offspring; (2) Chapter 4 - to determine the early cardiac changes in male and female fetuses exposed to maternal obesity; (3) Chapter 5 - A) to identify how the maternal environment is altered in obese pregnancy, and in particular if maternal obesity leads to hypoxic fetal conditions. B) To establish a maternal antioxidant (CoQ10) supplementation intervention during obese pregnancy; (4) Chapter 6 - to establish the effects of maternal exercise intervention during obese pregnancy on male and female fetal cardiac function. In this thesis it was shown in Chapter 3 that young adult mouse offspring of obese mothers had sex-specific mild cardiac systolic, diastolic and electrical dysfunction in vivo, which was more severe in male offspring. This was accompanied by a mild cardiac hypertrophy phenotype, with increased cell size in both male and female offspring of obese dams. The sex-specific differences in the functional and structural phenotypes of the 8-week-old offspring heart in response to maternal obesity may help to explain the different outcomes observed in male and female risk of developing cardiovascular disease. Chapter 4 shows that maternal obesity induced sex-specific changes in the lipidome of the fetal heart, with more changes observed in female fetuses, despite both sexes being exposed to the same maternal milieu. Changes in lipid supply to the fetal heart in obese pregnancies may drive the altered fetal cardiac transcriptome to promote a premature switch from glycolytic to β-oxidative metabolism in both sexes. The changes in lipid composition and metabolism were not accompanied by any alteration or premature maturation of fetal cardiac structure. These effects of maternal obesity on the fetal heart may contribute to the programming of altered cardiac structure and function in later life, though the precise mechanisms for this remain to be determined. In Chapter 5, it is shown that maternal obesity resulted in fetal, but not placental, hypoxia at e13.5 in both sexes. Fetal hypoxia was associated with increased maternal fat mass and hyperinsulinaemia in obese pregnancy. Alterations in either placental size and structure or fetal growth and metabolism did not appear to underlie the development of fetal hypoxia. Obesity induced maternal iron deficiency may contribute to the development of fetal hypoxia. This chapter also established a maternal CoQ10 supplementation intervention protocol in obese pregnancy, confirming supplementation had no effects on diet palatability or maternal appetite, and did not cause any adverse maternal or fetal outcomes. Chapter 6 showed that maternal obesity resulted in fetal growth restriction which was not rescued by maternal exercise intervention. Maternal obesity also reduced fetal insulin levels, which was sex-specifically rescued by maternal exercise in male fetuses only. Maternal obesity affected fetal in vivo cardiac function with altered heart structure and function which was not rescued by maternal exercise. These differences occurred in the absence of any changes in uterine or umbilical blood flow in response to either maternal obesity or exercise intervention. Overall, this thesis showed the effects of maternal obesity on the adult and fetal offspring heart in both males and females. It also determined the effects of maternal exercise intervention on the fetal heart and established an antioxidant intervention protocol. Maternal interventions in obese pregnancy are important for preventing the programming of cardiometabolic disease in offspring. Further studies to fully establish the potential short- and long-term implications of interventions, in both sexes, is important for identifying effective treatment strategies in obese pregnancies to protect the health of future generations.

In many populations worldwide over 50% of women have a body mass index above 25. Therefore, an increasing number of women enter pregnancy overweight or obese. This increases the risk for pregnancy complications such as Gestational Diabetes Mellitus (GDM). Placental function is often affected in obese and GDM pregnancies, which affects fetal growth and development. Recent years have implicated placental extracellular vesicles (EV) in fetal-maternal communication, with their microRNA (miRNA) content affecting maternal metabolism and potentially the fetus. Overall, it is well-established that obese and glucose-intolerant pregnancies have short- and long-term health consequences for mother and child. Women with GDM are therefore treated to control maternal glycaemia and thereby prevent effects on the fetus caused by excessive glucose exposure. In many countries metformin (an oral glucose-lowering agent) is now the first line pharmacological treatment for GDM as it can control maternal glycaemia and reduce gestational weight gain. However, metformin can cross the placenta and thereby directly affect the fetus. A few studies in humans and in animal models have reported increased adiposity in offspring exposed to metformin during pregnancy. However, information regarding the immediate actions of metformin on the placenta and fetus is limited. This thesis therefore aimed to assess the impact of metformin treatment of an obese and glucose-intolerant pregnancy in a mouse model on (i) the mother (described in chapter 3), (ii) fetal growth and placental structure (described in chapter 4) and (iii) on the placental lipidome and transcriptome (described in chapter 5). A last aim of this thesis was (iv) the establishment of isolation of placental EVs and characterisation of their miRNA content in an obese and glucose-intolerant pregnancy (described in chapter 6). In this thesis it is shown (chapter 3) that feeding mice a diet high in sugar and fat prior to and throughout pregnancy increased maternal fat mass and impaired glucose tolerance. Additionally, a preeclampsia-like phenotype with impaired uterine artery compliance and increased serum sFlt levels was induced. All these parameters were improved by treatment of the dams with metformin at clinically relevant doses prior to mating and throughout pregnancy. These findings are consistent with metformin being beneficial for maternal metabolic health, as observed in human studies, including recent data highlighting the potential of metformin to prevent preeclampsia. Chapter 4 highlights that placentas from obese dams had calcium depositions and a reduced labyrinthine zone. Calcium deposits have been observed previously in pregnancies complicated by GDM and obesity, showing that our model resembles the human situation. The obesity-induced placental pathologies together with the reduced uterine artery supply likely led to the fetal growth restriction observed. Metformin treatment did not rescue any of these obesity-induced changes in the fetus and the placenta. Metformin crossed the placenta and entered the fetal circulation at levels equivalent to maternal concentrations and was also taken up into fetal tissues. Metformin can therefore exert direct effects on the placenta and/or the fetus that may explain why fetal growth restriction and placental impairments are not rescued by metformin despite the improvement in uterine artery compliance. Reduced body weight in babies exposed to metformin in utero has previously been shown in human studies. Metformin did not affect placental AMPK and mTOR signalling as previously reported, but increased apoptosis markers in the male placenta. As the placenta accumulated substantial levels of metformin and is the key interface between mother and fetus we investigated in chapter 5 effects of metformin on the lipidome and transcriptome by comparing obese untreated and obese metformin-treated placentas. Metformin reduced triglycerides with low carbon numbers and free carnitine. A reduction of carnitine has previously been linked to preterm birth. Additionally, phosphatidylserines (PS) and sphingosine (18:0) were increased in the male placenta and lyso-phosphatidylcholine was reduced in the female placenta upon metformin treatment. A few human studies reported previously increased rates of preterm births in pregnancies exposed to metformin, thereby the reduced carnitine levels together with increased PS levels that could be linked to apoptosis in the male placenta warrant further investigation. Placental transcriptome analysis identified no major changes in mRNA expression upon metformin exposure but highlighted overall sex differences within the placenta that were consistent with increased in utero vulnerability of male fetuses to maternal obesity. Lastly, chapter 6 of this thesis shows the feasibility of extracting placental EVs released from placental explants. In a pilot study EVs and their miRNA cargo from male control placentas were compared to those from male obese placentas. miRNAs previously identified as playing a role in IGF2 signalling, mitochondrial dysfunction and preeclampsia were altered in obese placental EVs. Therefore, further follow-up could identify the role of placental EVs in matching maternal supply and fetal demand via modulating IGF2 signalling and could add evidence for their use as biomarkers to identify impaired placental function and preeclampsia. Overall, this thesis shows beneficial effects of metformin on maternal metabolic health and adds evidence to the current discussion of metformin as a preeclampsia treatment. However, it also highlights that metformin can have direct effects on the fetus and the placenta, with some evidence that at least some of these effects are sexually dimorphic. The use of metformin in pregnancy is complex and might be good for some women where beneficial effects of metformin in pregnancy might outweigh potential short-and long-term effects on the offspring. However, this might not be the case in other women. Therefore identifying treatment strategies/metformin formulations that retain maternal benefits whilst reducing fetal exposure should be the important goal of future studies.

Schizophrenia is a neurodevelopmental disorder (NDD) with debilitating symptoms, of which those in the cognitive domain (e.g. cognitive flexibility) are poorly treated by currently licensed medications. Exposure to viral, bacterial, or parasitic infections during pregnancy has been shown to increase risk for NDDs in offspring. This suggests that the effects of maternal immune activation (MIA) are due to inflammatory mediators, such as the pro-inflammatory cytokines interleukin (IL)-6 and tumour necrosis factor alpha (TNF-α). The postnatal maternal environment is also a critical period whereby maternal care behaviours promote normal development of the offspring brain. Impaired or attenuated maternal care in humans and rodents increases risk for NDDs in the adolescent and adult offspring. To understand the mechanistic pathways that link acute MIA with chronic NDDs in offspring, animal models are widely used. Such models commonly involve administration of immunogens into pregnant rodents, including the synthetic double-stranded RNA polyinosinic:polycytidylic acid (poly I:C) which mimics viral infection by binding to toll-like receptor 3. Whilst the poly I:C model has been used to investigate the contributions of the prenatal and postnatal maternal environments on offspring cognition in mice, this has not been investigated in rats, which offer significant benefits as a model system for complex cognitive and social behaviours. The experiments in this thesis involved systemic administration of poly I:C on gestational day (GD)-15 in Wistar rats to investigate effects on the early postnatal maternal environment and its contribution to the pathogenesis of cognitive deficits in offspring. The biomolecular characteristics of poly I:C (molecular weight, endotoxin contamination) were first investigated as a potential source of maternal and fetal phenotypic variability. Following this, a specified low molecular weight (LMW) and endotoxin-free poly I:C from InvivoGen was used. The effect of poly I:C on maternal weight (post-treatment and until GD21), the maternal pro-inflammatory response (plasma IL-6 and TNF-α at 3 h post-treatment), and litter composition was recorded. Pups were either left in the home litter or cross-fostered on postnatal day (PD)-1. During the early postnatal period (PD6-14), pup communicative behaviour (ultrasonic vocalisations, USVs) and maternal-offspring interactions were recorded. After weaning, female and male offspring were monitored for weight gain and tested on a battery of cognitive tasks (novel object recognition, NOR; elevated plus maze, EPM; social interaction, SI; radial arm maze, RAM; attentional set-shifting task, ASST) during adolescence (PD35), adulthood (PD100-125), or both. Offspring brain tissue was taken during adolescence and adulthood to investigate the effect of poly I:C on global DNA methylation, gene expression (Rln, Dnmt1, Pvalb, Sst, Gad1, Gad2, Slc6a1), and protein expression (GAD67, PVALB). Poly I:C induced a significant increase in maternal plasma TNF-α at 3 h post-treatment and reduced maternal weight gain from GD15-21. There was a variable effect of poly I:C on maternal weight loss at 6 and 24 h post-treatment and maternal behaviours. The prenatal maternal response to poly I:C programmed an increase in pup USVs and reduced postnatal weight gain. Similarly, prenatal poly I:C exposure programmed a significant and clinically-relevant deficit in cognitive flexibility in the ASST in adult offspring. This was observed in both females and males and was not found to be affected by alterations to the postnatal maternal environment. In the offspring brain, poly I:C induced a sex, postnatal age, and region-specific change in global DNA methylation (e.g. reduction in the PD35 female dorsal hippocampus, DHipp) and gene expression (e.g. reduced Rln in the PD175 DHipp), but had no effect on expression of GAD67 and PVALB protein in the adult offspring prefrontal cortex. Prenatal MIA using poly I:C provides a promising model for cognitive flexibility deficits in NDDs in which the efficacy of pharmacological and non-pharmacological therapeutic interventions can be assessed.

Ageing is a complex mechanism influenced by multiple factors. Stress, which could affect ageing, is mainly controlled by the hypothalamus-pituitary-adrenal (HPA)-axis and subsequent glucocorticoid (GC) release. Increases in GCs also induce long-lasting developmental changes in the organism (i.e. GC developmental programming), which may, in turn, affect brain ageing processes. This thesis aimed to determine the effects of stress on the brain by examining ageing biomarkers using both in-vitro and in-vivo approaches. In Chapter 2, the effects of stress biomarkers (GCs and alpha-amylase) were explored at cellular level, using HT22 (murine hippocampal neuronal cells). Within physiological concentrations, cortisol, but not corticosterone, showed a potential protective effect. Moreover, alpha-amylase protected cells against additional stressors, suggesting that increased stress biomarkers maintained cellular viability, instead of affecting senescent phenotypes. In Chapter 3, the typical mammalian brain ageing phenotypes were characterised in Japanese quail (Coturnix japonica) prior to investigating prenatal GC programming. Increased oxidative damage accumulation and changes in adult neurogenesis were found. Contrastingly, neuronal loss and increased white matter lesions were not observed. Chapter 4 investigated the prenatal GC programming effect in juvenile quail brains. Prenatal stress reduced glucocorticoid receptor expression in the amygdala and nidopallium, indicating prenatal GC programming; however, oxidative damage accumulation was unaffected. In Chapter 5, the prenatal GC programming effect was examined on both the stress response and neurogenesis in adult quails. Prenatal stress resulted in higher peak stress responses and increased neurogenesis, suggesting that it maintained high neuronal plasticity rather than enhancing typical ageing phenotypes. In conclusion, although ageing phenotypes are not necessarily conserved, comparative animal models can aid the investigation of human brain ageing. Stress may indirectly affect ageing via other systems. Therefore, investigating brain ageing at the system level should be considered as an alternative to focusing on single biomarkers in future.

Prenatal maternal immune activation (mIA) is associated with increased susceptibility to neurodevelopmental disorders (NDDs) in later life as evidenced by epidemiological studies that have associated prenatal infection with increased risk of NDDs such as schizophrenia (SZ). Prenatal induction of mIA in rodents produces behavioural and neuropathological phenotypes in the offspring, characteristic of NDD cognitive deficits. Previous studies in rodent models of mIA have characterised elicited maternal cytokine responses and altered cytokine balance in the placenta but are lacking with regards to how mIA affects functionality of placental and yolk sac nutrient transport systems. In this study it was hypothesised that mIA and the associated maternal proinflammatory cytokine response, induced by the viral mimetic poly(I:C), impairs placental amino acid transporter capacity, leading to altered fetal brain development and predisposition to NDDs later in life. This study administered poly(I:C) on gestational day (GD) 15 to pregnant Wistar dams to investigate the effects of mIA induction on maternal proinflammatory cytokine release and gestational growth trajectory, as well as characterising inflammatory changes and elicited effects in the fetoplacental unit, as compared to vehicle-treated controls. The molecular properties of poly(I:C) from two major commercial suppliers were found to differ with respect to molecular weight and endotoxin contamination, and this impacted on fetal phenotypic outcomes. Various techniques were employed to investigate the impact of poly(I:C)-mediated mIA induction on placental and yolk sac transport function, with a focus on system L amino acid transporters (LATs), including gene and protein expression analyses of LAT subunits in the placenta and yolk sac at 3 h (GD15), 24 h (GD16) and 6 days (GD21) following poly(I:C) administration and assessment of system L activity by measurement of in vivo maternofetal transfer of 14C-leucine at GD16 and GD21. The effect of poly(I:C) on fetal, placental and yolk sac growth and the concentration of branched amino acids (BCAAs) in these tissues, as well as placental morphology were investigated, and effects were examined according to fetal sex, to determine sexual dimorphic phenomena. At GD16, poly(I:C) induced sex-specific and gestational-dependent inflammatory responses in the placenta and yolk sac as well as on LAT expression and system L activity (reduced accumulation of 14C-leucine in the fetus), accompanied by reduced placental weight (in males) and disrupted labyrinth zone organisation. In contrast, at GD21, placental system L expression (but not yolk sac) and activity was increased in poly(I:C)-treated group, accompanied by an increased fetal plasma BCAA concentration (in males). Fetal bodyweight was unaffected by poly(I:C) treatment at all gestational timepoints, indicative of sustained fetal growth. However, fetal brain: bodyweight ratio was increased post-mIA at all timepoints. A biphasic response in the placenta to mIA characterised by an early impairment of system L transport (GD15, 16) followed by a compensatory upregulation of activity to maintain fetal growth (GD21) was observed. It is proposed that the earlier impairment of amino acid transport alters BCAA provision to the fetus, altering developmental trajectories of fetal organs and potentially influencing neurodevelopmental processes, further supported by decreased expression of SZ-susceptibility gene reelin in fetal brain 3 h post-poly(I:C) administration. This may predispose the fetus to NDD risk in postnatal life, despite the compensatory placental adaptation near term (upregulated system L transport) and maintained fetal growth. The pattern of LAT subtype expression differed between placenta and yolk sac, but supported the capacity for system La-mediated amino acid transport by both tissues. The ability of the rodent yolk sac to respond to mIA is presented for the first time and whilst responding similarly in the acute phase, it displayed divergent responses to the placenta at GD21. This may impact consequentially on a subset of vulnerable fetuses, reducing survival rates and detrimentally impacting on amino acid provision. In summary, poly(I:C)-mediated mIA evoked changes in placental and yolk sac system L amino acid transport function which could influence fetal amino acid provision, impacting on fetal brain developmental processes with enduring consequences, that could lead to NDDs in the offspring.

A strong body of evidence suggests that environmental insults from the point of fertilisation to birth and neonatal life can shape the health of the individual for many years to come. Adverse exposures, such as maternal overnutrition, in the early life environment increase the risk of traditionally adult-onset diseases such as cardiovascular disease and type 2 diabetes adding greatly to the next generation's burden of disease. Studies in animal models provide strong evidence that these effects are mediated by non-genetic programmed mechanisms. This is of particular concern, as recent studies in the UK suggest that over half of women are now overweight or obese during pregnancy. Current preventative strategies for adult cardiovascular disease have, thus far, focused on reducing an individual's modifiable risk factors. However, given growing evidence that risk of cardiovascular disease is determined in utero, there is strong rationale that disease risk from mother to child could be reduced prior to birth, through targeted interventions in the mother before and during pregnancy. Using an established murine model of maternal diet-induced obesity during pregnancy, the first aim of this thesis was to characterise potential programming factors in the obese mother and identify those that were targeted by a treadmill exercise intervention. Through feeding of an obesogenic diet, dams became heavier, with increased fat mass, and showed insulin resistance at weaning. Previous work has shown the intervention improved maternal insulin sensitivity during pregnancy (E19) and data from this thesis revealed that this was not accompanied by any changes to body composition. Previous data using this model showed that male offspring born to obese dams have pathological cardiac hypertrophy and ex vivo cardiac dysfunction. A second aim of this thesis was to establish if exercise intervention in obese dams was protective to the cardiovascular health of the offspring. These studies revealed that maternal exercise intervention during obese pregnancy had a positive impact by preventing pathological left ventricular cardiac hypertrophy and in vivo dysfunction, but did not prevent programmed hypertension in the male offspring. This demonstrates that offspring cardiac hypertrophy and dysfunction can be programmed independently of hypertension by maternal diet-induced obesity. The third aim of this thesis was to establish how female offspring were impacted by maternal obesity. The results demonstrated that female offspring born to obese dams were hypertensive and displayed right ventricular cardiac hypertrophy. However, there was no observable effect of maternal obesity on cardiac function in female offspring at this age. This highlights the potential sexually dimorphic effects of developmental programming by maternal obesity. A final aim of this thesis was to assess the immediate consequences of maternal obesity on the fetal heart and whether maternal exercise had any impact. This showed that in late gestation (E19), cardiac remodelling were already present in the male fetuses of obese dams, and the exercise intervention did not fully prevent this adverse finding. In conclusion, this thesis highlights that the cardiovascular health legacy of an individual is determined by maternal nutrition before birth and by the intrauterine environment. Just a small improvement in offspring risk could have important implications for the future prevalence of cardiovascular disease worldwide. Importantly this thesis highlights a potential need for combination intervention strategies to tackle the epidemic of obesity in pregnancy, as maternal exercise alone was not sufficient to reduce all aspects of the future burden of cardiovascular disease.

Maternal undernutrition during pregnancy can impair placental and fetal development and lead to programming of the fetus, with long-lasting effects such as an increased risk of disease later on in life including cardio-renal dysfunction and metabolic disorders. Fasting is a compulsory act for all adult Muslims during the holy month Ramadan, and although pregnant women are exempt from this practice, many still observe this religious requirement, abstaining from food or drink from dawn to sunset. The impact of prolonged maternal intermittent fasting (IF) on fetal development and offspring health is not well defined, but a reduced placental weight and birth weight have been reported previously. The aims of this study were to investigate the effects of IF during pregnancy on fetal development and offspring health and to examine sex-specific differences. A rat model was used to investigate the effects of maternal IF, mimicking the repeated daily fasting pattern of Ramadan fasting, to determine effects on 1) maternal physiology; 2) fetal development and metabolic profile; 3) placental growth, morphology and metabolomics; 4) placental system A amino acid transporter activity and expression; 5) long-term effects on postnatal growth and blood pressure; 6) pattern of feeding behaviour; 7) salt appetite and aversion; 8) cardio-renal function; and 9) response of offspring to postnatal salt dietary challenge. Food was withdrawn daily from pregnant Wistar rats from 5:00 pm to 9:00 am over 21 days of gestation; controls received food ad libitum and were used for comparison. Both groups had free access to water. IF dams consumed less food and gained significantly less weight, and at gestational day 21 (GD21) had reduced plasma glucose, glucagon and amino acid concentrations. At GD21, litter size was unaffected but IF fetuses of both sexes were significantly lighter and shorter with smaller head circumferences, but they demonstrated a higher brain:liver weight ratio. IF fetuses had normal plasma glucose and glucagon concentrations, but insulin and amino acid concentrations were decreased. Placental weight, and the junctional and labyrinth zone areas relative to total placental area were unchanged, but placental efficiency (fetal:placental weight ratio) was reduced in the IF group. The profile of placental metabolites was altered in the IF group, with sex-specific responses evident. Transplacental flux (measured as maternofetal clearance) of 14C-labelled α-methylaminoisobutyric acid (14C-MeAIB), a system A amino acid transporter substrate, was significantly reduced in both fetal sexes. However, measurement of system A activity as sodium-dependent 14C-MeAIB uptake into isolated placental plasma membrane vesicles was unchanged for both fetal sexes. The gene expression of system A transporter subtypes Slc38a1, Slc38a2 and Slc38a4 (encoding SNAT1, SNAT2 and SNAT4 respectively) was upregulated in the placentas of IF males, but was unaltered in placentas of IF female fetuses. However, no changes were observed in placental SNAT1 and SNAT2 protein expression for either sex in the IF group. At birth, IF neonatal weight was similar to control, but relative kidney weight in both IF sexes, and relative brain weight in IF females only, were reduced. Offspring of IF dams demonstrated altered postnatal growth and feeding behaviour, with IF females showing greater preference to salt intake. Blood pressure was similar between dietary groups at 5, 7 or 10 weeks of age with no changes in extracellular fluid volume. Glucose and insulin tolerance tests were unaltered by IF. In vivo renal 3H-inulin clearance was similar between both dietary groups at 14 weeks of age. However, challenging the IF offspring from week 5 of postnatal age with a high-salt diet induced high blood pressure in both sexes, with renal dysfunction evident in males at 14 weeks of age. In conclusion, a regimen of maternal IF, to model how nutrient intake may be perturbed during Ramadan fasting, was associated with several detrimental impacts on maternal physiology and fetal development, with changes also apparent in the profile of placental and fetal metabolites. Placental system A transporter activity measured in vivo was reduced and may contribute to the restriction of fetal growth seen in both sexes. Exposure to IF in utero had later consequences for the offspring, altering their growth trajectory and predisposing them to a higher risk of blood pressure and renal dysfunction in adulthood following a high-salt challenge, with sexual dimorphic responses clearly evident. Collectively, these observations suggest that Ramadan-type fasting during pregnancy, with repeated daily food deprivation, affects maternal and fetal health and that of the offspring in a sex-dependent manner, as well as placental function. Future refinements of the model to make it more translatable to human Ramadan fasting practice will provide further insights into the impact of Ramadan fasting during pregnancy and will empower pregnant Muslim women to make more informed decisions regarding their participation in the Ramadan fast.

Historically, sperm has been considered merely as a carrier of genetic material at fertilisation. However, it is known that sperm supports embryonic development better than other cell types, suggesting that it might also have additional important, non-genetic contributions to embryonic development. The work described in this dissertation focuses on identifying the molecular determinants of developmental programming of sperm. First, the development of embryos derived from sperm and spermatids, immature precursors of sperm was compared. Sperm-derived embryos developed significantly better than spermatid-derived embryos. Further research aiming to identify the reasons for the developmental advantage of sperm led to the identification of proteins that are present specifically in sperm and not in spermatids. Moreover, egg factors which are preferentially incorporated into the sperm, but not into the spermatid chromatin were identified with the use of egg extracts, suggesting that the chromatin of sperm could be programmed to interact with the components of the egg. Subsequently, the reasons for developmental failure of spermatid-derived embryos were investigated. By comparing the sperm with spermatids it was shown that the programming of sperm to support efficient development is linked to its special ability to regulate expression of developmentally-important embryonic genes, and not to its ability to support DNA replication or rRNA production. Further characterisation of the sperm and spermatid chromatin with the use of genome-wide sequencing allowed me to link the correct regulation of gene expression in the embryo with a certain combination of epigenetic marks in the sperm, but not in the spermatid chromatin. Finally, it is shown that enzymatic removal of epigenetic modifications at fertilisation leads to misregulation of gene expression. This therefore suggests that epigenetic information contained in parental genomes at fertilisation is required for a proper regulation of embryonic transcription. My results support the hypothesis that the sperm is not only a carrier of genetic material, but also provides the embryo with epigenetic information for regulation of transcription after fertilisation. I believe that these findings advance our current understanding of the nature and mechanisms of sperm programming for embryonic development, and are important contributions to the emerging field of transgenerational inheritance of epigenetic traits in general.

Much evidence indicates that maternal obesity programs a range of complications in offspring, highlighting the need to identify beneficial interventions. The present thesis examined if the programmed effects of maternal obesity on offspring are exacerbated by exposure to a high-fat, high-sugar ‘Cafeteria’ (Caf) diet and investigated the effects of a healthy diet intervention in adulthood. The first study examined whether a diet switch intervention could reverse the adverse effects of an unhealthy postweaning diet in male and female rat offspring born to dams fed standard chow or a high-fat, high-sugar, Caf diet. Weanling offspring from Caf dams were smaller and lighter, yet had more retroperitoneal (RP) fat, particularly in males. Maternal obesity enhanced the impact of postweaning Caf exposure on adult (14-week-old) body weight, RP fat, liver mass and plasma leptin in males but not females. Maternal Caf diet significantly increased relative expression of ACACA and Fasn in male and female weanling liver, whereas PPARα was increased in males from Caf dams. Hepatic CPT-1 expression was reduced in adult males from Caf fed dams. The results underline the sex-specific detrimental effects of maternal obesity on offspring; maternal obesity exacerbated the obesogenic phenotype produced by postweaning Caf diet in male, but not female offspring. A subset of rats from this first cohort were maintained on their postweaning diets until 22 weeks of age, when postweaning Caf groups were switched to chow for a further 5 weeks. Switching from Caf to chow in adulthood suppressed energy intake below groups maintained on chow. Consequently, body weight and adiposity fell in switched groups, but remained significantly higher than chow-fed controls. The diet switch improved a deficit in place recognition memory observed in Caf-fed groups, with no significant change over time in chow-fed groups. Importantly, the effects of the switch did not differ between offspring born to chow or Caf-fed dams. Thus, in these older adult offspring a healthy dietary intervention led to benefits regardless of prior exposure to maternal obesity. To complement these data, our second study investigated if the programming effects of maternal obesity were aggravated by offspring exposure to a Caf diet when introduced in adulthood. Male and female offspring from lean and obese dams were weaned onto chow until 9 weeks of age; siblings were then either continued on chow or switched to Caf diet for 5 weeks. Offspring from Caf dams were smaller than those from chow dams at birth, and exhibited greater adiposity, plasma glucose and leptin levels at weaning in both sexes. Offspring of Caf dams exhibited elevated liver triglyceride content at weaning but no significant changes in the liver antioxidant enzymes GPx, SOD, and CAT. The switch to Caf diet elevated body weight and fat mass, with more pronounced effects in females than males. As in our first study, adult males (14 week old) from Caf-fed dams exhibited increased body weight, adiposity, and plasma insulin and leptin levels relative to offspring from chow dams. In female offspring, only RP fat mass and plasma insulin were increased by maternal obesity. Moreover, there were behavioural effect of maternal obesity (reduced anxiety-like behaviour) on offspring. Thus, the effects of maternal Caf diet exposure were absent until a cafeteria diet challenge in adulthood, indicating that Caf diet-induced maternal obesity programs a latent vulnerability to obesogenic diet exposure in offspring, particularly in males. Further studies are needed to investigate the beneficial effects of reprogramming strategies such as healthy diet intervention in offspring, which are likely to be influenced by the duration, timing and mode of intervention.

The health impacts of prenatal Pb exposure can be framed with the Developmental Origins of Health and Disease (DOHaD) theory, which postulates that exposure to environmental factors (i.e., diet, chemicals, stress, etc.) during sensitive periods of development (i.e., pre-conception, gestation, neonatal, etc.) alters an organism’s molecular biology, physiology, and metabolism, potentially leading to myriad effects on cognition, growth, and maturation. Around the world, exposure to lead (Pb) continues to be a persistent problem. A particularly vulnerable population includes pregnant women, where Pb from both current and past exposures can affect their developing fetuses. Disruptions in normal epigenetic processes during gestation impact gene regulation and subsequent outcomes. Accumulating evidence for DOHaD includes potential mechanisms such as epigenetic reprogramming via DNA methylation (5mC) and, less studied, DNA hydroxymethylation (5hmC), linking developmental exposures to later-life outcomes. This dissertation aims to examine whether Pb exposure influences DNA 5mC and 5hmC and susceptibility to early cognitive delays. A human longitudinal cohort, Early Life Exposure in Mexico to Environmental Toxicants (ELEMENT), was used to investigate whether developmental Pb exposure would alter epigenetics at birth and in adolescence, and if these alterations are associated with decreases in neurodevelopmental scores in early infancy. The first aim of this dissertation characterized the association between prenatal Pb exposure measured in maternal blood and bone and CpG site-specific DNA methylation at >750,000 loci at birth in infant umbilical cord blood cells. Linear regression models were used to identify differentially methylated CpG sites by prenatal Pb exposure, using the false discovery rate method to correct for multiple testing. In the second aim, we evaluated both gene-specific DNA 5mC and 5hmC in adolescent whole blood from the same cohort to determine whether the association of prenatal Pb exposure and epigenetics persisted into adolescence and determine if 5hmC is associated with the exposure. Finally, in the third aim we assessed whether DNA methylation at birth mediates the association between prenatal Pb exposure and adverse neurodevelopmental outcomes at 12- and 24-months of age. In Aim 1, we found that trimester-specific developmental Pb exposures were associated with gene-specific DNA methylation changes at birth, and most changes were identified in genes related to neurodevelopment. Next, we showed that some of these Pb-induced DNA methylation changes were stable into adolescence, particularly those associated with first trimester exposure. This potentially suggests that perturbations occurring during the first trimester are reprogramming the epigenome and these changes remain into adolescence. Within Aim 2, we observed widespread, variable 5hmC levels in adolescent whole blood. We also revealed that prenatal Pb exposure has independent associations with both 5mC and 5hmC. These associations were also sex-specific at some loci, indicating a sexually dimorphic effect on the epigenome. Finally, the Aim 3 mediation analysis showed DNA methylation mediated the association between second trimester Pb exposure and cognition scores at 24-months of age at multiple neurodevelopmental measures. Overall, this dissertation illustrates that prenatal exposures to Pb can modulate epigenetics, and this may be one mechanism linking exposure to persistent effects on development and offspring health. Additional studies in larger cohorts or animal studies are needed in order to confirm these findings. These results emphasize the importance of distinguishing between 5mC and 5hmC during epigenetic studies, thus presenting a new conceivable mechanism describing the underlying biology behind the association between exposure and outcome, 5hmC

Accumulating evidence suggests that dysregulation of placental DNA methylation (DNAm) is a mechanism linking maternal weight during pregnancy to metabolic programming outcomes. The common marmoset, Callithrix jaccus, is a platyrrhine primate species that has provided much insight into studies of the primate placenta, maternal condition, and metabolic programming, yet the relationships between maternal weight and placental DNAm are unknown. Here, we report genome-wide DNAm from term marmoset placentas using reduced representation bisulfite sequencing. We identified 74 genes whose DNAm pattern is associated with maternal weight during gestation. These genes are predominantly involved in energy metabolism and homeostasis, including the regulation of glycolytic and lipid metabolic processes pathways.

Paternal obesity increases the risk for several metabolic diseases in offspring through developmental programming. Obesity induced oxidative stress is associated with sperm DNA damage, and this may partly contribute to the increased disease risk in offspring. We aimed to reduce oxidative stress induced sperm DNA damage in fathers by using a unique micronutrient supplement, designed to promote antioxidant activity, and prevent DNA damage. Specifically, we examined effects across various metabolic endpoints in fathers and offspring. We used a well-established model of diet-induced obesity where Sprague-Dawley (SD) rats were fed control or high fat (HFD) diet with or without supplement from weaning to 19 weeks of age, then they were mated with chow fed females. As expected, HFD increased body weight, adiposity, and fasting blood glucose, all of which were improved by dietary supplementation. Further metabolic investigations revealed that supplement ameliorated the hyperinsulinemia and reduced insulin sensitivity in HFD fed males. Hepatic lipids deposition was promoted by HFD, but reduced by the supplement, with altered expression of several genes involved in lipid metabolism. Furthermore, supplementation increased hepatic content of glutathione, a major antioxidant molecule. F1 offspring were weaned to chow or HFD at 3 weeks to investigate whether paternal HFD or supplementation would influence their response to postweaning HFD. In both male and female offspring, postweaning HFD increased body weight, adiposity, fasting blood glucose, glucose intolerance, fasting insulin and decreased insulin sensitivity. In male offspring, paternal HFD increased fasting blood glucose, which was lowered by paternal supplementation. Paternal supplementation improved insulin sensitivity in male offspring consuming postweaning HFD, whereas, female offspring from fathers fed control supplemented diet were more glucose intolerant when fed postweaning HFD. In summary, this thesis reports that a dietary micronutrient supplement had multiple benefits on the metabolic health of male rats. Furthermore, supplementation of fathers prior to mating improved glucose homeostasis in male offspring from HFD fed fathers, by reducing fasting blood glucose and improving insulin sensitivity.

Over the last 30 years, researchers have evaluated the various effects of feeding maternal high fat diets on the health of the offspring of various species, but mainly in rodents. Our hypothesis was that dietary source and amount of energy in maternal mouse diets would induce physiological, growth, and body composition changes in offspring independently of maternal body weight. The objective of this study was to evaluate the changes in body weight and composition from feeding maternal isocaloric diets on mouse offspring (1 generation). Parent mice were weaned and fed one of 3 diets consisting of either low fat with controlled calories based on prior ad libitum mouse experiment measurements (LFr), high fat diets restricted in calories to match the low fat diet (HFr), or high fat ad libitum (HFa) diets. Offspring from the first generation were fed either low fat ad libitum (LFa) or high fat ad libitum (HFa) diets and randomly assigned to four different dietary treatment groups: 1) dams fed LFr and offspring fed LFa (LFr:LFa) diets, 2) dams fed LFr and offspring fed HFa (LFr:HFa) diets, 3) dams fed HFr and offspring fed HFa (HFr:HFa) diets, and 4) dams fed HFa and offspring fed HFa (HFa:HFa) diets. Average weekly body weight and composition of offspring was determined for each of the four dietary treatments. Unfortunately, the experimental diets obtained from the manufacturer were severely deficient in all of the essential macro and micro minerals, which resulted in high pup mortality and prevented testing of the original hypothesis. Overall, both dams and offspring fed the LF diet had the greatest body weight gain and average percentage of body fat mass compared with those fed the other dietary treatments. In conclusion, feeding the mineral deficient diets confounded the effects of dietary calorie source and consumption on developmental programming in mice.

The objective of these 4 studies was to evaluate the effects of management decisions on reproductive performance of beef females. Experiment 1 evaluated the efficacy of a novel s.c. prostaglandin F2a injection on estrus synchronization and pregnancy success in yearling beef heifers. Heifers receiving a 2 mL s.c. injection of Lutalyse HighCon had similar estrus response and pregnancy rates compared to 5 mL Lutalyse i.m. within 2 different estrus synchronization programs. In experiment 2, May-calving heifers and primiparous cows were allotted to receive either no supplementation or supplement (0.45 or 0.91 kg/d per animal, heifers or primiparous cows, respectively) throughout the breeding season. Although supplementation increased BW, pregnancy rates were not impacted. In experiment 3, May-calving females were allotted to graze either sub-irrigated meadow or upland range throughout the breeding season. No differences in pregnancy rate were detected, despite differences in BW and BCS gain over the breeding season. Finally, experiment 4 examined the effects of varying levels of late gestation nutrition on dam and subsequent progeny performance. Multiparous, May-calving dams were allotted to graze either meadow or range forage and then to receive either no supplement or 0.45 kg/d per cow of a 33% CP supplement during late gestation. Prepartum meadow grazing tended to increase dam rebreed pregnancy rates. Heifer progeny had increased rebreed pregnancy rates as a primiparous cow, and steer progeny had increased marbling score if their dam grazed meadow. Dam supplementation increased BW of progeny over 2 generations. In summary, these experiments demonstrate the following findings: 1) a higher concentrate s.c. injection of prostaglandin F2a is effective in synchronizing estrus of beef heifers, 2) supplementation or differing forage type during the breeding season of a May-calving herd does not impact reproductive response, and 3) differences in late gestation nutrition of a May-calving herd results in altered progeny growth and performance. Advisors: Richard N. Funston and Jennifer R. Wood

Non-alcoholic fatty liver disease (NAFLD) is now the leading cause of chronic liver disease among youth in the United States. This recent rise of NAFLD may be partially due to perinatal programming, where in utero exposures alter the lifelong health trajectory of offspring. Maternal pregnancy diet and endocrine disrupting chemical exposure have been identified as drivers of perinatal programming. However, the potential for maternal diet to modify the impact of perinatal chemical exposure is not well understood. This dissertation examined whether perinatal exposure to two common environmental toxicants, bisphenol A (BPA) and high fat diets (HFDs), would affect NAFLD incidence in offspring. A longitudinal mouse exposure study and a human birth cohort were used to investigate this hypothesis and to evaluate the translation of findings across species. Oral exposure to one of six diets: Control, Western HFD, Mediterranean HFD or each diet with 50ug BPA/kg added, occurred pre-gestation through lactation. All mice were weaned onto the Control diet, thus isolating exposure to the perinatal period. Offspring NAFLD was assessed via hepatic steatosis and hepatic oxidative response at postnatal day 10 (PND10) and 10-months. Hepatic triglyceride (TG) levels were altered by perinatal HFD in dams, but in offspring perinatal exposures affected metabolic outcomes not hepatic TGs. Hepatic histology from 10-month offspring highly correlated with hepatic TG levels, validating the TG findings. Hepatic 8-isoprostane (8-iso) levels differed by perinatal exposure in PND10 and 10-month offspring, but alterations were age and sex-specific. Perinatal HFD and BPA minimally impacted offspring redox parameters (EhGSH, EhCys, S-glut), suggestive of greater homeostatic control of these parameters compared to lipid oxidation. Dam metabolic phenotype significantly altered offspring hepatic steatosis and oxidative response, even when perinatal HFD and BPA did not, emphasizing the critical role of the maternal environment on offspring health. The impact of maternal BPA exposure and gestational Mediterranean diet adherence (MDS) on the metabolic health of peripubertal youth was examined in a well-established human birth cohort. Youth metabolic and oxidative health was assessed via metabolic risk score (MRS) and serum 8-iso. Maternal pregnancy average and Trimester 2 BPA were associated with a suggestive decrease in youth MRS driven by boys, but a suggestive increase in 8-iso levels driven by girls. Maternal MDS did not affect youth MRS, but altered youth serum 8-iso in opposite directions based on sex. Additional youth characteristics (peripubertal BPA, MDS, vigorous activity, and pubertal status) contributed to predictive models of MRS and 8-iso, underscoring the impact healthy lifestyle behaviors may have, potentially even modifying perinatal programming. The unexpected lack of protection exerted by the Mediterranean diet in both mouse and human studies, suggests the beneficial effect observed in adults may not apply to perinatal exposure. Greater impact of HFDs in mice but BPA in humans highlights the need to carefully scrutinize findings before translating across species. Despite this difference, sex-specific effects occurred in both species, emphasizing the importance of investigating perinatal programming in all offspring. This research suggests that perinatal BPA and HFD exposure may be insufficient to induce perinatal programming of NAFLD. The significant impact of dam metabolic phenotype in mice and peripubertal behaviors in humans on metabolic and oxidative outcomes suggest NAFLD risk can be altered and potentially prevented at multiple life stages.

The Developmental Origins of Health and Disease hypothesis holds that during critical periods in development, organisms exhibit an enhanced plasticity enabling them to fine-tune their metabolism and patterns of gene expression in accordance with environmental cues. Such changes often confer immediate survival advantages, however, transient stresses experienced in utero may induce inappropriate adaptive changes that conflict with postnatal environments and consequently impair adult metabolic health. Preimplantation development has been recognized as a window of notable environmental sensitivity, and several animal studies have reported that nutritional, oxidative, and in vitro stresses restricted exclusively to this period are sufficient to alter developmental, growth and metabolic trajectories, leading to pathologies such as hypertension, dyslipidemia, and β-cell dysfunction in adulthood. This is particularly relevant to the over 5 million children conceived by in vitro fertilization (IVF), although it remains controversial whether in vitro embryo manipulation will have lasting effects on offspring health. Although IVF is considered safe, these children display modest changes in growth kinetics, fasting glucose levels, fat deposition and vascular function, which may signify a lasting and possibly dangerous effect of IVF on subsequent development and metabolic homeostasis.To address this controversy, we developed a mouse model to assess the long-term effects of IVF and found that even clinically optimized IVF conditions are sufficient to reprogram adult metabolism. In particular, female animals exhibit latent overgrowth, increased fat deposition and evidence of β-cell dysfunction, including impaired glucose-stimulated insulin secretion, basal hyperglycemia and insulinemia. Interestingly, males display no overt phenotype. Integrated microarray and metabolomics analyses of adult insulin-sensitive tissues identified sex- and tissue-specific IVF molecular signatures characterized by systemic oxidative stress, mitochondrial dysfunction, impaired insulin and adipogenic signaling, indicating that preimplantation stress can have unique and lasting developmental consequences. Additionally, female-specific increases in adipogenic and oxidative stress markers may explain the predisposition of IVF females to more severe phenotypes. Our data support the vulnerability of preimplantation embryos to environmental disturbance and demonstrate that conception by IVF can permanently reprogram growth trajectory and metabolic homeostasis through transcriptional and metabolic mechanisms with lasting consequences for adult growth and energy homeostasis. This has wide clinical relevance, underscoring the importance of continued follow-up of IVF offspring and future attention towards increasing the safety and efficacy of assisted conception.

Experiences, both in early life and adulthood, can profoundly modify brain and behavior in mammals. In the Long-Evans rat, two salient components of early life experiences are i) the quality of maternal care received and ii) environmental housing parameters/conditions, both of which are capable of modifying numerous components of the developing CNS, including stress or hypothalamic pituitary adrenal axis (HPA) functioning. The following studies address modifications to both brain and behavior (i.e. stress physiology and behavior, executive function, and reproduction) in Long-Evans rat offspring due to variation in the aforementioned parameters. Understanding how basic environmental variables are capable of influencing brain and behavior is important not just in the study of basic neurobiology but also for the exploration of potential sources of psychopathology. Section 2 of this dissertation provides a review of HPA axis regulation, while Sections 3 and 4 discuss relevant primate and rodent models of early life stress axis calibration. Section 6 addresses how early life maternal care is capable of differentially programing sexual behavior and the underlying hypothalamic pituitary gonadal axis (HPG). Section 7 focuses on early life maternal care and developmental programming of stress physiology and executive function. Finally, Section 8 investigates the role of housing parameters, specifically the role of animal bedding, in regulating stress sensitive behaviors. All the studies demonstrate the powerful effect of early life experience and developmental programming of brain and behavior in the rodent.

Recent advancements have uncovered environmental contributions to obesity and diabetes etiology. In fact, perinatal malnutrition resulting in low birth weight (LBW) has been shown to correlate with later life obesity and impaired glucose tolerance in aged offspring. LBW can result from a myriad of developmental perturbations including macronutrient restriction, hypoxia, maternal stress and toxin exposure. Polychlorinated biphenyls (PCBs) are ubiquitous environmental pollutants that bioaccumulate in the food chain resulting in dietary exposure in humans. Maternal and cord blood PCB levels are inversely associated with birth weight, and recent studies indicate that perinatal exposures to PCBs contribute to gender-specific obesity development in children. PCBs have also been shown to enter breast milk resulting in direct exposure in early postnatal life. Therefore, we hypothesized that perinatal PCB exposure cause developmental blight resulting in decreased birth weight and increased adiposity and glucose intolerance with aging. We found that mice perinatally exposed to PCBs did not differ in birth weight, but exhibited sex-specific effects on adiposity. Females perinatally exposed to PCBs were significantly more obese at 7 weeks of age while male offspring exhibited no difference in fat mass but had decreased lean mass compared to controls. With aging, the differences in females dissipated while the male offspring decreased lean mass persisted. Male offspring perinatally exposed to PCBs displayed impaired glucose tolerance at 7 weeks of age but normalized over time, while the females were glucose intolerant only after 6 months of age. This impairment of glucose tolerance was not attributed to insulin resistance. These data illustrate time-dependent and sex-specific perturbations of maternal PCB exposure on offspring body composition and glucose homeostasis. As the liver is a major facilitator in glucose homeostasis and xenobiotic detoxification, we investigated PCB-induced alterations in hepatic gene expression and found attenuated expression of glycolytic genes and increased expression of detoxifying and antioxidant genes in both PCB-exposed maternal and offspring livers. Taken together, these data demonstrate a role for perinatal pollutant exposure in the etiology of glucose intolerance. Further studies are required to elucidate the mechanisms causing sex-specific modulation of body composition and glucose intolerance.