Adverse maternal events around the time of conception influence embryonic development. Thus, aberrations in the uterine environment during early pregnancy, such as those resulting from maternal metabolic or nutritional disruption, can alter gene expression in the developing embryo, leading to variations in its developmental trajectory. Dietary supplementation of long-chain omega-3 polyunsaturated fatty acids (LCPUFA), especially Docosahexaenoic acid (DHA) improves metabolic and reproductive health across species. The objective of this study was to evaluate the effects of peri-conceptual LCPUFA supplementation on endometrial gene expression, uterine health and embryonic gene expression in overweight horses. Thirteen non-lactating light horse mares (mean ± SEM age=13.56±0.11 yr; mean ± SEM BCS=7.07±0.21) were supplemented with concentrate (n=6) or an isocaloric diet containing 0.06 g/kg BW algae-derived omega-3 LCPUFA (n=7) beginning 60 d prior to sample collection. Four consecutive ovulatory cycles were monitored, and uterine endometrial samples were obtained 12 d post-ovulation in cycles 1, 3 and 4. Mares were bred and embryos were flushed 12 d post ovulation 2,3 and 4. Endometrial biopsies obtained from supplemented mares contained increased DHA and omega-3 fatty acids as a percent of total fat (P< 0.05). Endometrial biopsy scores were assigned to endometrial tissues and mares receiving the LCPUFA supplementation had improved scores during the first ovulatory period as compared to control animals (P=0.009). Candidate genes essential to inflammation, prostaglandin synthesis and embryonic development were evaluated by quantitative reverse transcriptase polymerase chain reaction. Data were log transformed and analyzed using the GLM procedure in SAS (v9.3). When examining the data independent of breeding and pregnancy status, endometrial obtained samples from LCPUFA supplemented mares contained reduced IL6 (P= 0.04) and TNFa (P=0.03) mRNA abundance and tended to have increased transcript abundance for Uterocalin (P= 0.09), SAA (P= 0.06) and IL10 (P= 0.06). Endometrial samples from mares fed LCPUFA pregnant in cycle 3 contained greater IL10 (P< 0.001), PTGFS (P=0.05), OXTR (P=0.05) and PLA2G3 mRNA (P= 0.009) and had a tendency for increased SAA (P= 0.08), PTGES (P=0.10) and SLCO2A1 (P=0.10) mRNA abundance. Supplemented mares bred but not pregnant at day 12 in cycle 3 had reduced expression of PTGER2 (P=0.001) and PTGS1 (P= <0.001) in endometrial samples. In embryos obtained post ovulatory cycle 3 and 4, relative transcript abundance of GATA4 and GATA6, markers of endoderm differentiation, along with GATA3 and ELF3, markers of trophectoderm differentiation were greater (P< 0.05) in embryos from LCPUFA supplemented mares (n=5), than controls (n=5). These results indicate that algae-derived LCPUFA supplementation during the peri-conceptual period alters the post-ovulatory uterine environment in the horse by modifying expression of genes related to inflammation and regulating prostaglandin synthesis. Additionally, embryos obtained from supplemented mares displayed differential gene expression related to embryonic lineage specification. / Ph. D.

Renal blood flow and micropuncture studies were conducted in late gestation fetal sheep (gestational age 134 - 141 days; term 150 days) and neonatal lambs (8 - 18 days after birth) to study the forces involved in glomerular filtration (GFR) and characterize the tubuloglomerular feedback (TGF) system during development. These studies required the kidney to be immobilized so stable models in acutely prepared anaesthetized animals were developed. Fetuses were studied in a heated water bath exteriorized from the uterus but with an intact umbilical circulation. The lower GFR in fetuses than lambs was found to be due to both lower net filtration pressures (P<0.001) and a lower ultrafiltration coefficient (P<0.001). TGF was present at both ages, but in fetuses the sensitivity was higher (P<0.001) and reactivity was lower (P<0.001). The reduction in TGF sensitivity between fetal and neonatal life may facilitate the increase in renal blood flow and GFR which occurs at this time. In both fetuses and lambs the sensitivity of the TGF curve was reduced by volume expansion (P<0.001, P<0.05) and reactivity was reduced in lambs (P<0.001). Furosemide abolished TGF at both ages. In both fetuses and lambs, TGF reactivity was increased by inhibition of neuronal nitric oxide synthase (nNOS; P<0.01, P<0.001) and in lambs, TGF sensitivity was increased (P<0.01). This indicates that nitric oxide produced by the macula densa modulates TGF during development. In offspring destined to become hypertensive due to maternal dexamethasone treatment in early gestation TGF sensitivity tended to be enhanced in fetal life and was enhanced in lambs (P<0.01). Increased TGF sensitivity may contribute to the development of hypertension in this model of developmental programming. The effects of nNOS inhibition were attenuated in these animals, suggesting that they have low tonic production of nitric oxide by the macula densa. In fetuses whose mothers had been subtotally nephrectomized prior to mating to induce maternal mild renal impairment, GFR was increased (P<0.01) but net filtration pressure was reduced (P<0.001) so the ultrafiltration coefficient was increased (P<0.001). TGF sensitivity was normal and the effects of nNOS inhibition were similar to normal fetuses.

Renal blood flow and micropuncture studies were conducted in late gestation fetal sheep (gestational age 134 - 141 days; term 150 days) and neonatal lambs (8 - 18 days after birth) to study the forces involved in glomerular filtration (GFR) and characterize the tubuloglomerular feedback (TGF) system during development. These studies required the kidney to be immobilized so stable models in acutely prepared anaesthetized animals were developed. Fetuses were studied in a heated water bath exteriorized from the uterus but with an intact umbilical circulation. The lower GFR in fetuses than lambs was found to be due to both lower net filtration pressures (P<0.001) and a lower ultrafiltration coefficient (P<0.001). TGF was present at both ages, but in fetuses the sensitivity was higher (P<0.001) and reactivity was lower (P<0.001). The reduction in TGF sensitivity between fetal and neonatal life may facilitate the increase in renal blood flow and GFR which occurs at this time. In both fetuses and lambs the sensitivity of the TGF curve was reduced by volume expansion (P<0.001, P<0.05) and reactivity was reduced in lambs (P<0.001). Furosemide abolished TGF at both ages. In both fetuses and lambs, TGF reactivity was increased by inhibition of neuronal nitric oxide synthase (nNOS; P<0.01, P<0.001) and in lambs, TGF sensitivity was increased (P<0.01). This indicates that nitric oxide produced by the macula densa modulates TGF during development. In offspring destined to become hypertensive due to maternal dexamethasone treatment in early gestation TGF sensitivity tended to be enhanced in fetal life and was enhanced in lambs (P<0.01). Increased TGF sensitivity may contribute to the development of hypertension in this model of developmental programming. The effects of nNOS inhibition were attenuated in these animals, suggesting that they have low tonic production of nitric oxide by the macula densa. In fetuses whose mothers had been subtotally nephrectomized prior to mating to induce maternal mild renal impairment, GFR was increased (P<0.01) but net filtration pressure was reduced (P<0.001) so the ultrafiltration coefficient was increased (P<0.001). TGF sensitivity was normal and the effects of nNOS inhibition were similar to normal fetuses.

High vitamin (HV), AIN-93G diet with ten-fold the regular amount of vitamins (RV), consumed by pregnant Wistar rats increases characteristics of metabolic syndrome (MetS) in their first litters. Therefore, the effects of the maternal HV-diet on tissue mechanisms regulating insulin resistance in offspring (Part 1) and on characteristics of MetS in the dams and their second litter (L2) offspring (Part 2) were examined. Part 1 (studies 1 and 2) hypothesis was that the maternal HV-diet alters tissue fatty acid (FA) concentrations, expression of peroxisome-proliferator-activated receptors (PPARs) genes, and their regulation of metabolism in the offspring, favoring insulin resistance. Part 2 (studies 3 and 4) hypothesis was that high-vitamin intakes during the first pregnancy increase weight gain, food intake and markers of MetS in both the dams and their litters. In all experiments, dams were fed the RV or HV-diet. In study 4, a high-folic-acid-diet (HFol, RV+10-fold folic acid) was added. In studies 1 and 2, the offspring were weaned to an obesogenic diet. The HV-diet affected tissue FA concentrations (study 1), increased muscle PPAR-ɑ mRNA levels and uncoupled relationships between hepatic PPAR-γ mRNA levels and insulin resistance (study 2) in male offspring. In study 3, dams fed the HV-diet during the first pregnancy were maintained on the RV-diet and then mated again after 12wk. Their litters were fed the RV-diet. The HV-diet increased weight gain and food intake of both dams and L2, and insulin resistance in their offspring. In study 4, both HV and HFol-diets increased post-weaning weight gain, but differed in their effects on biomarkers of food intake regulation. In conclusion, feeding the HV-diet during the first pregnancy increases post-weaning body weight and food intake in Wistar rat dams, uncouples tissue regulation of glucose metabolism and promotes characteristics of MetS in their litters. Folic acid is not the only vitamin involved.

High vitamin (HV), AIN-93G diet with ten-fold the regular amount of vitamins (RV), consumed by pregnant Wistar rats increases characteristics of metabolic syndrome (MetS) in their first litters. Therefore, the effects of the maternal HV-diet on tissue mechanisms regulating insulin resistance in offspring (Part 1) and on characteristics of MetS in the dams and their second litter (L2) offspring (Part 2) were examined. Part 1 (studies 1 and 2) hypothesis was that the maternal HV-diet alters tissue fatty acid (FA) concentrations, expression of peroxisome-proliferator-activated receptors (PPARs) genes, and their regulation of metabolism in the offspring, favoring insulin resistance. Part 2 (studies 3 and 4) hypothesis was that high-vitamin intakes during the first pregnancy increase weight gain, food intake and markers of MetS in both the dams and their litters. In all experiments, dams were fed the RV or HV-diet. In study 4, a high-folic-acid-diet (HFol, RV+10-fold folic acid) was added. In studies 1 and 2, the offspring were weaned to an obesogenic diet. The HV-diet affected tissue FA concentrations (study 1), increased muscle PPAR-ɑ mRNA levels and uncoupled relationships between hepatic PPAR-γ mRNA levels and insulin resistance (study 2) in male offspring. In study 3, dams fed the HV-diet during the first pregnancy were maintained on the RV-diet and then mated again after 12wk. Their litters were fed the RV-diet. The HV-diet increased weight gain and food intake of both dams and L2, and insulin resistance in their offspring. In study 4, both HV and HFol-diets increased post-weaning weight gain, but differed in their effects on biomarkers of food intake regulation. In conclusion, feeding the HV-diet during the first pregnancy increases post-weaning body weight and food intake in Wistar rat dams, uncouples tissue regulation of glucose metabolism and promotes characteristics of MetS in their litters. Folic acid is not the only vitamin involved.

Fetal exposure to excess glucocorticoid is associated with low birth weight and increased cardiovascular disease risk in first generation offspring. Such phenotypes can be produced experimentally through the administration of the synthetic glucocorticoid dexamethasone (Dex) to pregnant rats during the last week of gestation. These ‘programmed effects’ can be transmitted to a second generation through both maternal and paternal lines. The overall hypothesis for this thesis was that the transmission of programmed effects through the male line may result from alterations in fetal germ cells, which form sperm in adulthood. Epigenetic reprogramming of germ cells is characterised by the genome-wide erasure and subsequent re-establishment of 5-methylcytosine (5mC), however this process has not previously been described for the rat. Furthermore, the involvement of more recently identified cytosine modifications; 5-hydroxymethylcytosine (5hmC), 5- formylcytosine (5fC) and 5-carboxylcytosine (5caC), has not been characterised during germ cell ontogeny. Using immunofluorescence to study DNA modifications during late gestation I identified that 5hmC, 5fC and 5caC were present between e14.5 and e16.5 but absent thereafter. In contrast, 5mC was absent during this time but remethylation was noted from e19.5 onwards. Prenatal Dex exposure was associated with the presence of significantly more 5mC-positive germ cells at e19.5 relative to controls. This difference did not persist at e20.5 suggesting that Dex exposure promotes premature global remethylation. The mechanisms for this are unclear since there were no differences between groups in the localisation of the DNA methyltransferases DNMT3a and 3b, or in markers of normal testis maturation. To enable the study of gene-specific changes in DNA methylation in the germline a colony of Germ Cell Specific-Enhanced Green Fluorescent Protein (GCS-EGFP) rats was established and characterised. GCS-EGFP rats had a transgenerational decrease in pup weight with Dex exposure, as in Wistar rats. The expression of both established and novel candidate genes was compared between strains. Multiple genes across different pathways had altered expression, with some affected in both Wistar and GCS-EGFP rats, whilst other differences were strain-specific. Enhanced Reduced Representation Bisulfite Sequencing was performed on liver and fetal germ cells from males exposed to Dex in utero to explore effects on DNA methylation. These studies confirm that epigenetic reprogramming occurs in the rat and that this process may be susceptible to modification by prenatal Dex exposure. GCS-EGFP rats also exhibited a Dex programming phenotype, with decreased pup weight and altered liver gene expression. The use of this unique strain of rats will permit dissection of the mechanisms for the transmission of programmed phenotypes across generations.

Americans consume three percent more total daily calories from sugar than current recommendations. Maternal diets high in sugar can cause obesity and diabetes mellitus. Objectives were to compare supplemental dietary sucrose to a protein alternative on maternal health and fetal programming utilizing a sow biomedical model. Pregnant sows (Landrace × Yorkshire, average BW = 222 ± 35 kg, n = 21) were fed a corn-soybean meal-based diet (CSM) at one percent BW at 0700 h daily from d 29 (± 1.47) to 111 (± 0.58) of gestation. Sows were randomly assigned to dietary supplement treatments: 126 g CSM (CON, n = 5), 110 g cooked ground beef (BEEF, n = 6), 85.5 g sucrose (SUCR, n = 5), or the combination of 54.8 g BEEF and 42.7 g SUCR (B+S, n = 5). Dietary supplements were fed three times daily from d 40 to 110 (± 0.58) of gestation. A repeated measures design was modeled using the MIXED procedure of SAS. Dietary treatment did not influence gestational BW (P ≥ 0.99), subcutaneous fat depth (P ≥ 0.09), blood chemistry panel (P ≥ 0.21), or total-, HDL-, or LDL-cholesterol, triglyceride, insulin, or C-reactive protein serum concentrations (P ≥ 0.07). Dietary treatment did not influence sow organ or lean tissue weight (P ≥ 0.42). Compared to CON, BEEF fetuses had increased BW (P = 0.01), crown to rump length (P = 0.01), nose to crown length (P < 0.01), heart girth (P = 0.02), and abdominal girth (P = 0.05). Dietary treatment did not influence fetal growth characteristics of median weight male and female fetuses (P ≥ 0.23). Compared to BEEF, SUCR fetuses had heavier liver weights (P = 0.04). Dietary treatment by sex interaction occurred for fetal kidney weight with BEEF males having heavier kidney weights compared BEEF females (P = 0.03). Dietary treatment did not influence other fetal organ or lean tissue weights (P ≥ 0.09). These results suggest beef or sucrose supplementation at 1.49 or 1.16 grams per kilogram BW per day, respectively, from day 40 to 110 of gestation had minimal impact on maternal health and fetal development. / North Dakota Beef Commission; Topigs Norsvin; and North Dakota State University Agricultural Experiment Station

Diagnostic radiation is typically avoided during pregnancy, as the effect of low-dose radiation exposure on the fetus is uncertain. The objective of this study was to determine if ionizing radiation exposure during late fetal development would cause an adverse intrauterine environment, and lead to growth restriction of offspring and a hypertensive phenotype later in life. To study this, pregnant BALB/cJ mice were exposed to ionizing radiation at 5, 10, 50, 100, 300 or 1000 mGy on gestational day 15. Offspring were weighed weekly from the age of weaning until a mature age of 16 weeks. Cardiovascular effects were assessed every other week via heart rate and blood pressure measurements using tail plethysmography. The expression of genetic markers for endothelial dysfunction, inflammation, mitochondrial capacity, and regulation of the oxidative stress response in the aorta and heart for the 1000 mGy was assessed from tissue collected at 17 weeks of age. We observed no effects of low to mid dose (5-300 mGy) radiation on offspring growth and blood pressure. Growth restriction was observed in male and female offspring exposed to high-dose radiation (1000 mGy). In the heart, there was no observed effect on mitochondrial capacity and oxidative stress response genes. In the aorta, we observed decreased TNF-α expression in male offspring, which may be linked to the growth restriction but was not considered a sign of cardiovascular dysfunction. There were no observed effects of exposure to 1000 mGy on cardiovascular function. This study provides knowledge on the possible effects of radiation on in utero development, which broadens the knowledge on the range of stressors capable of affecting offspring growth and development. / Thesis / Master of Science (MSc)

Imprinted genes are a subset of loci, positioned on autosomes and the X-chromosome, which are expressed monoallelically in a parent-of-origin specific manner. The influence of such genes on the regulation of embryonic growth and postnatal energy homeostasis is well established. The parental conflict hypothesis predicts that, in utero, paternally-expressed genes will promote maternal resource acquisition and thus growth, whereas maternally-expressed genes will oppose this action, restricting resource investment in a single brood in the interests of the lifetime reproductive success of the mother. Grb10 is an imprinted gene which encodes the cytoplasmic adaptor protein Growth factor receptor bound protein 10. In the majority of tissues, Grb10 is expressed from the maternally-derived chromosome. Consistent with conflict theory, transgenic mice inheriting a disrupted Grb10 allele through the maternal line (Grb10Δ2-4m/+) exhibit embryonic overgrowth, although the mechanisms and signalling pathways responsible for this effect are unclear. Grb10Δ2-4m/+ mice also demonstrate enhanced insulin signalling and improved whole body glucose clearance, consistent with the established role of imprinted genes in the regulation of postnatal metabolism. An integrated LacZ gene-trap in the Grb10Δ2-4 allele failed to fully recapitulate endogenous Grb10 expression, notably within the central nervous system. To address this issue, a second transgenic mouse line, Grb10KO, was created. This allele produced strong LacZ reporter expression in the central nervous system, but only when transmitted through the paternal line (Grb10KO+/p), establishing Grb10 as the only known imprinted gene with a reciprocal imprinting profile between the central nervous system and peripheral tissues. Grb10KO+/p mice exhibit a social dominance phenotype, suggesting distinct roles for maternally- and paternally-expressed Grb10, consistent with their respective sites of expression. The current study characterised the Grb10KO allele at the genetic level, and in doing so, revealed a phenotypic difference between Grb10KOm/p and Grb10Δ2-4m/p mice for which a possible explanation was provided. Importantly, with this knowledge, the current study elucidated the genetic and molecular basis for inconsistencies in reporter expression between the two transgenic lines, identifying a novel tissue-specific enhancer element at the locus. In addition to the central nervous system, this enhancer appeared to be active in the mammary epithelium, identifying a novel site of Grb10 expression, which was pregnancy-dependent and specifically from the maternally-inherited chromosome. Characterisation of the functional significance of expression in this tissue revealed that maternally-expressed Grb10 mediates a supply/demand system between lactating mother and suckling pup, acting as a supply promoter and demand suppressor. This role is inconsistent with conflict theory, but suggests the maintenance of the Grb10 imprint in the mammary epithelium might be associated with improved coadaptiveness between mother and offspring. Intriguingly, in utero, Grb10 is both a demand and supply suppressor. When considered together, these findings suggest a wider role for maternally-expressed Grb10 in the homeostatic control of growth and achievement of optimal fitness.

<p>Environmental stress during embryonic development could lead to growth restriction of the embryo, and act as a risk factor for the development of cardiovascular disease in adult life. A common environmental stressor that causes growth restriction is prenatal hypoxia, which has been shown to adversely affect adult health in mammalian models. Prenatal hypoxia causes an increase in catecholamines which results in over stimulation of the cardiac β-adrenergic receptors. Previous work on chickens has shown that prenatal hypoxia causes an increase in the sensitivity of β-adrenergic receptors to epinephrine in the embryonic heart. The sensitivity of these receptors was found to be decreased in prenatal hypoxic juvenile. Prenatal hypoxia has no significant effect on the density of these receptors in neither the embryo nor the juvenile. The lack of change in receptor density implies that the effects of hypoxia are further down stream in the signalling cascade. The β2 adrenergic receptor can couple to both the stimulatory Gα subunit (Gsα) and the inhibitory Gα subunit (Giα). We hypothesized that prenatal hypoxia would cause an increase in the Gsα in the sensitized embryos, while increasing Giα in the desensitized juveniles. This study evaluated the relative levels of Gsα and Giα in the hypoxic chicken embryo, and in the prenatally hypoxic juvenile, Using western blotting. Hypoxia considerably increased Giα in the chicken embryo while having no effect on Gsα. In the prenatally hypoxic juvenile Gsα was significantly increased while no changes were found in Giα. This dissociation between the levels of Gα subunit and receptor sensitivity implies that that hypoxia affects the signaling cascade downstream of the Gα subunit.</p>