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.

A link between early malnutrition and development of components of the metabolic syndrome later in life has been shown in epidemiological and animal data. Moreover, studies now tend to demonstrate that not only fetal environment is important for developmental programming but postnatal milieu could also participate to this process. The “predictive adaptive response” hypothesis stipulates that not only a suboptimal environment during fetal life will lead to development of metabolic disorders later in life but more likely is a mismatch between the early environment and that one really encountered later on that increases the risk of developing later disease. Based on this hypothesis, we examined the effect of an early mismatched environment produced by fetal protein restriction and postnatal catch-up growth on the development of obesity and cardiovascular disease in male mice. We focussed our study on the analysis of adipose tissue with in vitro examination of differentiation, proliferation of preadipocytes. We also investigated in vivo the development of overweight in adult mice and we measured the expression of specific adipose tissue molecules with microarray. Finally, we investigated the development of hypertension and atherosclerosis in parallel to obesity. Our results indicated that postnatal catch-up growth after fetal protein restriction favours the development of obesiy in adult male mice. Early mismatched nutrition also influenced the capacity of proliferation of preadipocyte as well as the expression of adipose tissue specific molecules involved mainly in lipid biosynthesis. Finally, early nutrition also induced hypertension in adult male mice while no influence of fetal protein restriction and postnatal catch-up growth was observed on atherosclerosis development.

Maternal and fetal stressors during development can permanently alter various physiological functions and impact long-term health. These alterations are said to be programmed because they persist long after the original insult. Current evidence indicates that iron deficiency (ID) during pregnancy can induce a host of long-term programming effects, including cardiovascular complications and behavioural deficits. Despite the relevance of ID as a model of developmental programming, these effects have not been extensively studied. The purpose of the present series of experiments was to develop a model of maternal ID throughout pregnancy to study its long-term cardiovascular and behavioural consequences in neonatal and adult offspring. Female rats were fed either a low iron diet (3 mg/kg or 10 mg/kg Fe) or a control diet (> 225 mg/kg Fe) prior to and throughout gestation. At birth, all dams were fed a control diet (270 mg/kg Fe). This treatment caused altered growth trajectories which persisted in adulthood. Adult perinatal ID (PID) offspring, despite showing no signs of anemia at that time, had persistent elevations in arterial pressure (AP), as well as enhanced responsiveness of AP to high and low sodium intake. These animals also had altered responsiveness of renal medullary blood flow to changes in AP. PID offspring also had altered function of intrarenal and vascular nitric oxide signaling. Similar studies performed in acute ID animals revealed opposite trends in intrarenal and vascular NOS function, as well as in effects on the cardiovascular system. In addition to the cardiovascular effects, adult PID male offspring exhibited a number of behavioural changes, as assessed by monitoring locomotor activity in their home cages (by radiotelemetry) as well as in a novel environment. PID male offspring also performed poorly in a Morris water maze compared to controls. These differences were not observed in female PID animals. In summary, these studies provide evidence that ID during gestation has deleterious effects on various aspects of the offspring’s physiology. Given the global incidence of ID, as well as its propensity to afflict pregnant women, developmental programming from this condition could have profound implications on global health. / Thesis (Ph.D, Pharmacology & Toxicology) -- Queen's University, 2009-01-22 23:32:40.931

The thrifty phenotype hypothesis proposes reduced nutrition alters the trajectory of development of metabolic regulatory systems to produce a phenotype better fitted to an environment of decreased later-life nutrient availability. Because organisms have physiological mechanisms for coping with poor nutrition, they may have sociobehavioral mechanisms as well. Aggressive behavior, especially in the context of feeding competition, may be advantageous in such environments. There could be an association between aggression and intrauterine growth restriction (IUGR), which can result from low maternal food intake during pregnancy. The main hypotheses of this study are [1] IUGR offspring demonstrate higher rates of aggressive behavior and [2] IUGR offspring attain higher ranks. Behavioral observations were conducted on 22 juvenile baboons (Papio sp., ages 3-5 yrs) living in groups. Male IUGR (n = 4) and female IUGR (n = 5) were offspring of mothers fed 70% the same feed eaten by control (CTR) mothers in pregnancy and lactation. CTR males (n = 8) and CTR females (n = 5) were offspring of mothers fed ad libitum. Some authorities recommend this moderate level of dietary restriction for health and longevity. Offspring have not experienced dietary restriction since weaning. IUGR, compared to CTR, showed significantly increased rates of aggressive behavior, especially threat displays. Differences were more dramatic in males than in females. IUGR baboons performed the affiliative display behaviors lipsmack and chatter at elevated rates too, perhaps to counteract the effects of increased aggressive displays. IUGR females exhibited increased rates of stereotypical chewing behavior, while IUGR males exhibited decreased rates of play behavior, possibly indicating elevated anxiety levels. There was only limited support for condition-based differences in rank. Elevated rates of aggression in IUGR baboons may reflect an aggressive behavioral phenotype that enhances fitness by improving access to resources. Alternatively, they could be a non-adaptive result of neurodevelopment with a potentially negative impact on fitness. Unraveling the dynamic relationship between experiences and development is essential for understanding how phenotypes are formed. This will improve the ability of mothers to assess benefits of different nutritional strategies, leading to healthier individuals not just during growth and development, but throughout life.

Chronic prenatal hypoxia is associated with intrauterine growth retardation and there is now some evidence that it also induces programmed hypertension in offspring. However these studies are generally confounded as hypoxia is either induced by maternal hypoxia or placental insufficiency. The study described in this thesis is designed to overcome this problem. Pregnant rats were dosed daily with the drug dofetilide (2.5 mg/kg) or water on GD 11-14 and the cardiovascular parameters of the offspring at 8-12 weeks (>300g) were analysed using implanted telemetry blood pressure (BP) transmitters.Dofetilide is a class III antiarrhythmic drug that selectively blocks the Ikr channel which is expressed in the rat embryo but not in the adult rat. When administered to pregnant rats it induces bradycardia (and associated hypoxia) in the embryos without affecting maternal oxygenation or heart rate. Embryo culture studies showed that dofetilide induced a period of embryonic bradycardia for up to 9 hours following each dose. The dofetilide treated-rats had less completed pregnancies, smaller litters and lower weight pups compared to controls. At 8-12 weeks age the dofetilide offspring has increased BP (+10-12%) compared with controls. Postnatal stress in the form of air puffs did not reveal other cardiovascular differences between control and dofetilide offspring. The increased BP was not associated with an increased HR or changes in the autonomic nervous system. Remaining unexplored possibilities include impaired nephrogenesis, vascular dysfunction and microvascular

The circadian rhythms are not solely regulated by photoperiod but are also influenced by feed regimen. Therefore, maternal nutrient restriction during gestation could potentially impact the fetal circadian rhythm. Melatonin, a circadian rhythm modulator hormone, has shown to act as an antioxidant reducing reactive oxygen species during stress exposure; and two potential mechanisms have been proposed for melatonin causing vasoconstriction and vasodilation regulating blood flow. In livestock species, nutrient restriction during gestation reduces uterine blood flow, limiting nutrients availability to the fetus for growth and development. Therefore, this project aimed to use beef heifers to evaluate the maternal nutrient restriction and/ or melatonin supplementation in (1) temporal transcript abundance of clock genes, angiogenic factors and nutrient sensing genes in bovine placenta, (2) temporal alterations of uteroplacental blood flow, vaginal temperatures, and placentome vascularization, and (3) fetal morphometrics. Early maternal nutrient restriction did not alter placental explants gene expression. Furthermore, the maternal portion of the placentome exhibited limit temporal variation, while the fetal tissue exhibited a clear temporal rhythm in the mRNA relative abundance of the genes measured. Additionally, melatonin supplementation during late gestation, showed to increased uterine blood flow, reduced vaginal temperatures, and rescued fetal weights during compromised pregnancies in a season dependent manner. In conclusion, the bovine placenta exhibited an autonomous circadian rhythm, while the fetal and maternal circadian rhythms appeared to be independent systems. Future research should examine the effects of melatonin supplementation in fetal organ development.

Protein restriction during rodent pregnancy is a well-established model of developmental programming. Although the Southampton low-protein diet model of developmental programming has been accepted to produce hypertensive offspring, the mechanism of this programming remains unclear. Currently the effects of protein restriction in the Southampton low-protein diet are confounded by a relative elevation of the amino acid methionine. The aim of this project was thus to clarify the roles of protein reduction and methionine elevation within this model, especially within the context of amino acid and one-carbon metabolism. Pregnant Wistar rats were fed casein-based diets ad libitum varying in casein (18% or 9%) and methionine content (0.5% or 1.0%) from day 0 through 20 of pregnancy. Two diets exactly replicated the Southampton control and low-protein diets (Con: 18% casein, 0.5% methionine; LP: 9% casein, 0.5% methionine), while a third low-protein high-methionine diet (LP-MET: 9% casein, 1.0% methionine) was employed as a positive control for methionine stress. On day 20 of gestation dams were sacrificed and the feto-placental unit was excised; maternal and fetal blood was collected for HPLC analysis of free amino acids. Maternal plasma was also analyzed for homocysteine content using a spectrophotometric-based enzyme assay. Diet did not affect maternal weight gain, food consumption, litter size or fetal weight. In dams and fetuses, methionine was significantly elevated in both low-protein groups. Maternal homocysteine was significantly elevated only in dams fed the low-protein high-methionine diet. Reductions in maternal serine, proline and glycine levels also occurred in dams fed the low-protein high-methionine diet; fetuses of these dams had significantly reduced levels of all three branch chain amino acids (leucine, isoleucine and valine). Both low-protein diets resulted in drastic reductions in circulating threonine levels in dams and fetuses. Thus, ingestion of low-protein diets with a relative (0.5%) or overt excess (1.0%) of methionine appears to disrupt one-carbon metabolism at the level of homocysteine remethylation to methionine. This may place strain on the folate cycle, as may be indicated by the reduced levels of threonine, serine and glycine. Further testing is necessary to clarify the extent to which folate stores are being utilized for homocysteine remethylation. Increased competition for placental amino acid transport may explain the alternations in circulating free fetal amino acids. Further investigations into levels of other one-carbon metabolites in dams and fetuses are necessary to fully characterize the effect of low-protein high-methionine diets, particularly within the context of the Southampton model of developmental programming.

Protein restriction during rodent pregnancy is a well-established model of developmental programming. Although the Southampton low-protein diet model of developmental programming has been accepted to produce hypertensive offspring, the mechanism of this programming remains unclear. Currently the effects of protein restriction in the Southampton low-protein diet are confounded by a relative elevation of the amino acid methionine. The aim of this project was thus to clarify the roles of protein reduction and methionine elevation within this model, especially within the context of amino acid and one-carbon metabolism. Pregnant Wistar rats were fed casein-based diets ad libitum varying in casein (18% or 9%) and methionine content (0.5% or 1.0%) from day 0 through 20 of pregnancy. Two diets exactly replicated the Southampton control and low-protein diets (Con: 18% casein, 0.5% methionine; LP: 9% casein, 0.5% methionine), while a third low-protein high-methionine diet (LP-MET: 9% casein, 1.0% methionine) was employed as a positive control for methionine stress. On day 20 of gestation dams were sacrificed and the feto-placental unit was excised; maternal and fetal blood was collected for HPLC analysis of free amino acids. Maternal plasma was also analyzed for homocysteine content using a spectrophotometric-based enzyme assay. Diet did not affect maternal weight gain, food consumption, litter size or fetal weight. In dams and fetuses, methionine was significantly elevated in both low-protein groups. Maternal homocysteine was significantly elevated only in dams fed the low-protein high-methionine diet. Reductions in maternal serine, proline and glycine levels also occurred in dams fed the low-protein high-methionine diet; fetuses of these dams had significantly reduced levels of all three branch chain amino acids (leucine, isoleucine and valine). Both low-protein diets resulted in drastic reductions in circulating threonine levels in dams and fetuses. Thus, ingestion of low-protein diets with a relative (0.5%) or overt excess (1.0%) of methionine appears to disrupt one-carbon metabolism at the level of homocysteine remethylation to methionine. This may place strain on the folate cycle, as may be indicated by the reduced levels of threonine, serine and glycine. Further testing is necessary to clarify the extent to which folate stores are being utilized for homocysteine remethylation. Increased competition for placental amino acid transport may explain the alternations in circulating free fetal amino acids. Further investigations into levels of other one-carbon metabolites in dams and fetuses are necessary to fully characterize the effect of low-protein high-methionine diets, particularly within the context of the Southampton model of developmental programming.

: L’économie de la filière équine repose aujourd’hui sur la production de chevaux athlètes performants sur le long terme. Le métabolisme de la jument gestante peut programmer le développement du poulain, sa santé à long terme et donc ses performances sportives à l’âge adulte. De nombreuses pratiques d’élevage peuvent modifier le métabolisme maternel, telles que la nutrition durant la gestation, la surnutrition durant la vie de la jument (surpoids et obésité) et le nombre de poulains produits par la jument (parité). L’objectif de ce travail était d’étudier les effets du métabolisme maternel durant la gestation sur la fonction et la structure placentaire à terme, la croissance osseuse, le métabolisme énergétique, l’inflammation systémique et le statut ostéoarticulaire des poulains en croissance. Un premier modèle de perturbation nutritionnelle en fin de gestation a été développé en comparant des juments ayant ingéré uniquement des fourrages au cours de la gestation ou bien des fourrages et des concentrés à partir de la mi-gestation. Ce modèle a permis de montrer que la supplémentation en concentrés altérait le métabolisme glucidique maternel, la fonction placentaire ainsi que le statut ostéoarticulaire et la réponse métabolique à un challenge de surnutrition chez le poulain. D’autre part, une perte d’état trop importante associée à une qualité/quantité de foin insuffisante entrainait un retard de maturité des fonctions de métabolisme énergétique et de reproduction mâle chez les poulains. Un deuxième modèle a ensuite été développé pour étudier l’effet de la primiparité. Cette étude a confirmé que la croissance fœtale des poulains issus des juments primipares était réduite et que ces poulains demeuraient plus petits avec un métabolisme glucidique et une maturation testiculaire retardés par rapport aux poulains issus de juments multipares. Le troisième modèle développé s’est intéressé à l’effet de l’obésité maternelle dès la conception. En effet, la prévalence de surpoids et d’obésité est de plus en plus importante au sein de la filière équine. Ce dernier modèle a permis de montrer que l’obésité maternelle associée à une résistance à l’insuline et une inflammation systémique augmentées entrainait une augmentation de la résistance à l’insuline, de l’inflammation systémique et du développement de lésions d’ostéochondrose chez les poulains. L'ensemble de ces résultats met en avant la relation entre la résistance à l’insuline maternelle, l’inflammation maternelle et le développement de lésions d’ostéochondrose chez les poulains durant la croissance, mais également entre sous-nutrition utérine et retard de maturité. Ces observations vont permettre de développer de nouvelles recommandations nutritionnelles pour les poulinières. / The economy of the equine industry is based on the production of high performance athlete horses. The metabolism of the pregnant mare can program the development of the foal, its long-term health and therefore its athletic performance at adulthood. Many breeding practices can modify maternal metabolism, such as nutrition during pregnancy, overnutrition during the mare's productive life (overweight and obesity) and the number of foals produced by the mare (parity). The aim of this work was to study the effects of maternal metabolism during pregnancy on placental function and structure, as well as bone growth, energy metabolism, systemic inflammation and osteoarticular status in growing foals. In a first approach, mares fed with forage only during gestation were compared to mares fed forage and concentrates from mid-gestation. Supplementation with concentrates altered maternal carbohydrate metabolism and placental function. In weaned foals, the osteoarticular status and the metabolic response to an overnutrition were also affected by the use of concentrates in maternal nutrition. Conversely, mares fed forage only lost body condition, which led to a delay in the post-natal maturation in terms of energy metabolism and testicular function in foals. In a second approach, the effect of primiparity was studied. Foals born to primiparous mares were growth restricted at birth and had a long-term maturational delay in bone growth, carbohydrate metabolism and testicular function. Finally, given the current high prevalence of overweight and obesity in the equine species, the effects of maternal obesity were studied. Maternal obesity associated with increased maternal insulin resistance and systemic inflammation resulted in increased insulin resistance, systemic inflammation, and increased incidence of osteochondrosis in foals. Altogether, these results highlight the relationship between maternal insulin resistance, maternal inflammation and the development of osteochondrosis lesions in foals during growth, but also between in utero undernutrition and maturation delay. These observations will contribute to adjust nutritional recommendations to broodmares.

Approximately 10% of pregnant women take antidepressants. Prenatal exposure to selective serotonin reuptake inhibitors (SSRIs), a class of antidepressants, has been shown to alter serotonergic signaling in the brain. However, the effects of SSRIs on peripheral serotonin (5HT) synthesis and/or signaling have largely been ignored. Serotonin in the gut is critical for intestinal function and dysregulation of this pathway is associated with intestinal disease. Therefore, the goal of this study was to determine the effects of perinatal exposure to the SSRI fluoxetine (Prozac®) on intestinal health in the offspring. Dams were given vehicle or fluoxetine hydrochloride (FLX 10 mg/kg/d; N=15) for 2 weeks prior to mating until weaning. We assessed markers of serotonergic signaling, inflammation, and composition of the gut microbiota in the offspring. Male offspring of fluoxetine-treated dams had significantly elevated serum levels of 5-HT and decreased expression of the 5HT2A receptor and MAO. In female offspring there was no effect of SSRI exposure to alter any components of serotonergic signaling. Although we did not find any evidence of increased inflammation following fluoxetine exposure, there were significant alterations in the composition of the gut microbiota in the exposed offspring. Male offspring of SSRIs-exposed mothers had changes in key components of the gut serotonergic system in association with elevated levels of serum 5-HT and alterations in the gut microbiota in adulthood. The impact of these changes on intestinal health and the reasons for the sex specific effects remain to be determined. / Thesis / Master of Science in Medical Sciences (MSMS)