Perinatal asphyxia is thought to be a major cause of subsequent neurological deficits. Pathological studies suggest that many of these events occur before birth. However, the relationship between specific prenatal events and neurological outcome is not clear. This thesis tested the hypothesis that certain fetal factors play a role in determining the severity and distribution of neuronal loss following in utero asphyxia. Chronically instrumented fetal sheep at three different gestational ages; midgestation (90d), late-gestation (120-130d) or near term (> 135d) were subjected to either a single or repeated insult. The insult consisted of an episode of either systemic asphyxia (umbilical cord occlusion) or cerebral ischaemia (carotid artery occlusion). The fetal parietal cortical electroencephalogram (EEG), cortical impedance (CI) indicating intracellular edema, blood pressure (MAP), electrocardiogram (ECG) and frequent fetal blood gases and metabolites were measured. Three days after the insult, histopathological analysis or immunohistochemistry was performed to determine neuronal loss and specific neurotransmitters respectively. Transient (10min) occlusion of the umbilical cord in late-gestation fetuses, resulted in severe fetal asphyxia, hypotension (24±5mmHg, p<0.01), bradycardia (72±14bpm, p<0.001), depressed EEG activity (-17±2dB, p<0.001) and intracellular edema. The intracellular edema resolved within 27±6min, whereas the EEG activity was depressed for 5±2h, despite rapid recovery of pO2. Neither seizures or infarction were observed. The degree of hypotension, increase in CI, lactate and recovery of post-asphyxial EEG intensity were more marked in 135d fetuses compared with the midgestation fetus (p<0.01). Neuronal loss, which was only observed in the older group, was predominantly in the hippocampus and associated with the severity of hypotension during occlusion. Repeated episodes of cerebral ischaemia, altered the distribution of neuronal loss compared with single insults, inducing damage mainly in the striatum. The frequency of the insults determined the severity of the damage. Similarly, recurrent episodes of fetal asphyxia induced predominantly striatal neuronal loss. Each occlusion resulted in fetal hypoxia and bradycardia accompanied by increased T/QRS ratio as noted on the ECG. Progressively severe hypotension and lactic acidosis developed during successive occlusions. The EEG was depressed and CI increased with each occlusion. After the asphyxial episodes, blood pressure and heart rate returned to normal, while the T wave was inverted for 310±60min. The EEG remained depressed for 90±10min and intermittent seizures developed at 3.3±0.6h after the last occlusion. The extent of neuronal loss correlated with the degree of hypotension, increase in T/QRS ratio, duration of post-asphyxial EEG depression and number of seizures. Immunohistochemical analysis showed loss of striatal GABAergic projection neurons. These findings demonstrate that certain prenatal factors, such as neurological maturation, pattern of the insult and cardiovascular instability can influence neuronal outcome following fetal asphyxia. An isolated brief episode of asphyxia can lead to selective hippocampal neuronal loss, while repeated insults induce predominantly striatal damage. These distributions of neuronal loss may be associated with postnatal sequelae such as learning disorders and cerebral palsy.

Lactose malabsorption (LM) in children was diagnosed by an elevated breath hydrogen (BH) level following a 10g lactose load. A portable gas chromatograph and a semiconductor detector, designed and constructed for use in the Pacific Islands is described. Following verification on known malabsorber and. normal subjects, the technique was used to determine the prevalence of LM in Europeans at Auckland and Rarotonga, and in Samoans at two locations in Auckland and two locations in Western Samoa. The prevalence of LM in Europeans was significantly (p<0.01) higher at Rarotonga than at Auckland. For Samoans, the prevalence of LM was significantly (p<0.01) higher in Western Samoa than at Auckland. The prevalence of LM was very highly significantly (p<0.001) related to race. Each child tested for LM filled in a questionaire to determine attitude, consumption of and perceived intolerance to milk, milk biscuits and ice cream. Lactose malabsorption was significantly (p<0.05) correlated to milk consumption and to attitude to dairy products, but not to sex, age, and perceived intolerance. The consumption of dairy products was very highly significantly (p<0.0001) correlated to attitude, and highly significantly (p<0.001) correlated to location and perceived intolerance. There was no significant correlation between consumption and race, sex or age. Perceived intolerance to individual dairy products was significantly correlated to attitude to milk (p<0.0001), milk biscuits (p<0.02) and ice cream (p<0.001). Perceived intolerance was not related to age, sex, race, location or the actual symptoms following the consumption of 10g lactose. The unexpected finding of increased LM in the Pacific Islands, was investigated further by studying the LM status of the Medical Team during a visit to western Samoa, and by performing a microbiological survey of water quality. It was found that half of the Medical Team 3/6, became malabsorbers during the week spent in Western Samoa. On returning to New Zealand it was shown that lactase levels took 3 months to normalise. The water supply in Western Samoa was shown to contain very high levels of coliform bacteria. The currently held hypothesis that genetic factors are solely involved in the onset of LM, was not supported. The evidence from the survey supported environmental factors are also involved in adult onset LM. The hypothesis suggesting that dietary lactose was a requirement for retaining elevated lactase levels, was tested using Galactosemic and Phenyl Ketonuria patients. None of the patients had developed LM although they had been on a low lactose diet for years, hence the theory was not supported. The BH method proved highly successful in diagnosing LM with many of the children actually enjoying it.

In newborn infants, birth asphyxia represents the predominant cause of brain injury. These infants will later exhibit neurodevelopmental disabilities or a more major cerebral palsy. Prevention of adverse outcomes requires an understanding of the way in which these deficits develop. Endogenous protective mechanisms arising from the insult have opened new insights in neuroprotective strategies. Neurotrophic factors such as IGF-1 and its N-terminal tripeptide GPE have been shown to confer some neuroprotection after HI injury in the adult rodent. In the P21 rat brain after moderate HI injury, exogenous intracerebral and intraperitoneal injections of GPE (30μg and 300μg respectively) were neuroprotective in the hippocampus and lateral cortex possibly through binding to glia as detected by autoradiography of 3H-GPE. In the preterm infant the mechanisms of white matter injury remain to be clearly elucidated. To mimic the pattern of diffuse cerebral injury of the very preterm infant, a transient moderate focal HI injury has been applied on the immature P3 rat. This new model showed a significant reduction in the lateral cortical volume with reduction and alteration of the myelination pattern in the cortical white matter (WM) at P21. These cortical alterations result from neuro-axonal damage 24h after the insult as shown with Fluoro-Jade B staining and β-APP accumulation. In addition activated astrocytes from 24h after HI up to P21 were present. This model should enable us to elucidate some of the pathogenic mechanisms involved in diffuse WM injury. Brain damage in the developing brain has two components: 1) the pattern and mechanisms of injury are correlated with the stage of development at the time of injury; 2) it will influence subsequent brain development.

Ovine placental lactogen (oPL) is secreted by the placenta into both the fetal and maternal compartments. Its biological function(s) during pregnancy and the mechanisms involved are still unclear. A purification procedure was developed for oPL from sheep placental cotyledons of late gestation. Four procedures were attempted to obtain homogeneous oPL. Recoveries of oPL and total protein were measured throughout the several procedures using a specific oPL RIA and the Bradford protein estimation respectively. The third and fourth procedures resulted in homogeneous oPL and a partial amino acid sequence was obtained from the fourth procedure. In the successful procedures, the placental tissue was extracted with 0.1 M ammonium bicarbonate pH 8.5. A pH precipitation of the soluble fraction was performed, followed by 60% saturation with ammonium sulphate. Further separation steps involved chromatogaphic procedures. Carboxymethylcellulose (CM32) cation exchange was performed batchwise at pH 5.6. Subsequently chromatofocusing was performed to elute proteins in order of their isoelectric points. This was carried out using a pH gadient of 0.9 to 6.0. The final chromatographic step was reverse-phase high performance liquid chromatography (RP-HPLC) using a C4 column. To obtain homogeneous oPL in the third procedure, the partially purified oPL was subjected to SDS polyacrylamide gel electrophoresis and the separated proteins were transferred to nitrocellulose membrane. The homogeneous oPL was eluted from the membrane, however, sequencing was unsuccessful. It was assumed that the N terminal of oPL was blocked. Homogeneous oPL was obtained in the fourth procedure by electrophoretic elution from the Hunkapiller gel system performed at 4°C. The oPL was digested with trypsin, the fragments were separated by RP-HPLC chromatography and two peptides were sequenced. Peptide 1: F D E Q Y G Q G I Peptide 2: Y I N C H T Several strategies were attempted to provide more homogeneous oPL to enable more sequencing. The partially purified oPL fractions from each of these attempts were pooled and electrophoresed on an SDS polyacrylamide gel. The section of acrylamide containing the oPL band was homogenised and a trypsin digest was performed. The digested oPL was separated from the gel pieces, filtered through a Sep-Pak filter and the fragments were separated by RP-HPLC. The yield of oPL was low, but sufficient homogeneous oPL was obtained to provide a partial amino acid sequence from tryptic peptides. A further two peptides provided sequences. Peptide 3: (L) A G E M V N R F D E Q Y G Q G I Peptide 4: (L) Q P G K C Q I P L Q S L F Collaborators from Genentech Inc (San Francisco USA) used partially purified oPL produced from the present study and also obtained homogeneous oPL (Colosi et al., 1989). Complementary DNA clones of oPL were isolated and expressed in mammalian cells by recombinant DNA techniques (Colosi et al., 1989). These clones were sequenced, demonstrating that the full sequence of oPL consists of 198 amino acids preceded by a 38 amino acid sequence signal. Recombinant oPL was generated by Colosi et al. (1989) which provided sufficient material to perform physiological studies in vivo. The somatogenic effects of recombinant oPL were investigated in the growth hormone (GH) deficient dwarf rat and compared to identical doses of recombinant bovine GH (bGH) in 3 independent studies. Both oPL and bGH treatments resulted in an increase (p<0.05) in body weight gain compared to that in saline treated controls, with oPL treatment being more potent than bGH (p<0.05). In promoting linear growth, oPL was more potent (p<0.05) than bGH in some instances. Nitrogen content of dry carcass matter was increased with oPL treatment compared to saline (p<0.05), with a nonsignificant increase in bGH treated animals. Carcass fat was similarly reduced by both oPL and bGH treatment (p<0.05) compared to saline. Serum insulin-like growth factor I (IGF-I) concentrations were increased significantly (p<0.05) by both oPL and bGH treatments, with a significantly greater effect of oPL suggested in one study. No increase in hepatic IGF-I mRNA was evident with either treatment, suggesting that the increase in serum IGF-I is due to posttranscriptional mechanisms. The expression of IGF binding protein 3 (IGFBP-3) hepatic mRNA was increased (p<0.05) with bGH treatment compared to that after saline treatment, but was unaffected by opL treatment, indicating regulation by GH at the transcriptional level. The binding of [125I]bGH to hepatic membrane preparations demonstrated no difference in specific binding compared to that in saline controls. However, [125I]oPL specific binding was greater in oPL treated animals (p<0.05). Animals treated with bGH had reduced (p<0.05) hepatic GH receptor mRNA compared to saline controls, but oPL treatment had no effect. Thus, oPL is a potent anabolic and lipolytic agent in the dwarf rat, exerting greater somatogenic effects on some parameters than bGH. The studies in this thesis have described biochemical and biological characterisitics of oPL. The amino acid sequence of oPL is more closely related to prolactin (PRL) than to GH (Colosi et al., 1989). However, oPL has potent somatogenic activities in the GH deficient dwarf rat. Our data suggest differences in receptor binding and effects on GH receptor and IGFBP-3 expression with these two treatments, raising the possibility of actions through different pathways or differential effects at the GH receptor level. These results do not fully resolve whether GH and PRL exert all effects through a single receptor or whether there is a separate PL receptor.

The introduction to this thesis is a literature review. Kramer, in a study commissioned by WHO, reviewed the literature prior to 1985 on low birthweight. This is extended, mainly in respect to infants who are small for gestational age with emphasis on important findings in relation to birthweight since that time. Work in New Zealand on birthweight is also summarised. The literature is also reviewed in respect to the mechanisms in the pathway between the placenta and the fetus, and in respect to recent work suggesting a link between birthweight and disease in adult life. This thesis examines factors that influence birthweight and placental weight. Birthweight for gestational age percentile curves for the New Zealand population were firstly defined. small for gestational age (SGA) infants could then be categorised. The thesis considers two sources of data, the first a cross-sectional sample of the New Zealand population from 1987 to 1990 (the control subjects of the New Zealand cot Death study, a national case-control study on sudden infant death syndrome), and the second a hospital population in Auckland (National Womens Hospital (l992)). These two datasets are investigated to determine factors that influence birthweight in a univariate situation and then in the multivariate situation. Independent variables are considered using a priori categorisations and where appropriate Quantile-Quantile (Q-Q) derived categorisations determined by producing plots of the quantiles of cases versus controls. A number of variables under the headings of socio-demographic, lifestyle, genetic, obstetric and nutrition are examined and found to be associated with the outcomes of interest at the univariate level. After controlling in multivariate analyses a number of variables are found to be no longer significant, however some show strong relationships. The variable relating to smoking in both datasets shows the greatest detrimental effect on the outcomes considered in respect to birthweight. This confirms that in New Zealand, as in other places in the world, smoking has significant consequences on birthweight. The data is also investigated for the timing of insult to the fetus from smoking, and is found to be most important during pregnancy. comparison of the results comparing those obtained using a binary outcome for SGA, and those obtained using birthweight continuously, show relatively consistent results. The odds ratios and the decreases in birthweight obtained from both datasets show a relatively linear relationship between the two. An examination into whether a distinct group of individuals exists in respect to having large placentae for birthweight, indentified an artefact in the dataset relating to recording of placental weight for twins. After removal of twins from the dataset, examination of factors that influence placental weight showed that the factors that influence placental weight are not the same as those that influence birthweight. In particular smoking is found not to influence placental weight, and haemoglobin, which has no influence on birthweight, is found to be inversely associated with placental weight. other factors such as parity are found to influence placental weight in the same proportion in which birthweight is affected. In conclusion this thesis shows that factors investigated in New Zealand are consistent with findings in the international literature in relation to birthweight. The results on factors that influence placental weight add to the international literature on a topic on which little work has been carried out. The results of this thesis point to areas where future research needs to be carried out, in particular in relation to maternal nutrition during pregnancy and maternal energy expenditure during pregnancy. There is also a need for further research into the relationships of factors on placental weight and the ratio of birthweight to placental weight, and how these relationships affect health outcomes in childhood and adult life.

Over the past decade, a number of epidemiological studies have provided significant evidence that certain major adult noncommunicable diseases, such as hypertension, ischaemic heart disease and non-insulin dependent diabetes mellitus, may be associated with impaired fetal growth. This phenomenon has been termed "programming" which is essentially the term used for persisting changes in structure and function caused by undernutrition or other adverse influences acting during critical periods of early development. Programming has been used as the mechanistic basis to explain the long term sequelae of intrauterine growth retardation (IUGR). The mechanisms underlying the epidemiological observations remain to be elucidated and developed. While it is well established that severe maternal undernutrition during pregnancy leads to IUGR, there has been relatively little well defined animal studies of the somatotrophic axis and postnatal development of growth retarded offspring. The major objectives of this thesis were to establish a model in the rat of IUGR by nutritional restriction of the dam throughout gestation and to examine the effects of fetal growth retardation on endocrine, molecular and growth parameters during postnatal development. In addition, the development of an animal model for IUGR enabled well defined studies testing distinct hypotheses suggested by the epidemiological observations of professor David Barker and colleagues. Timed matings were performed in Wistar rats and dams were randomly assigned to one of two dietary treatment groups. A control group was fed ad libitum throughout pregnancy and a restricted group was fed 30% of ad libitum intake. Restricted fed dams were observed to lose a significant amount of body weight throughout gestation, due to undernutrition, but caught up to the ad libitum group during the lactating period. Maternal undernutrition significantly reduced fetal and placental weights without altering litter size. Postnatally, body weights of offspring from undernourished dams continued to be reduced until at least 18 weeks of age, although they were observed to be growing at the same rate as ad libitum offspring by 2 weeks of age. A cohort of animals from undernourished dams were maintained to measure blood pressure by tail cuff plethysmography. Offspring from undernourished dams were found to have significantly elevated systolic blood pressures from 18 weeks of age. This observation provides direct experimental support for the hypothesis, derived from human epidemiological studies, that the origin of adult hypertension may originate during fetal life as a result of exposure to a sub-optimal intrauterine environment. Parallel reductions in plasma IGF-I and hepatic IGF-I mRNA concentrations before 15 days of age were also observed in growth retarded offspring. Hepatic IGF-I transcription start sites within exon 1 and exon 2 were coordinately reduced with IUGR up to 15 days of age without changes in GHR and GHBP mRNA abundance. The lack of catch-up growth observed in the IUGR offspring despite normalization of their plasma IGF-I and IGF-I mRNA levels from 15 days of age may be due to a state of partial resistance to GH. This observation lead to a series of treatment studies in which neonatal and juvenile offspring from ad libitum and undernourished dams were treated with growth factors to investigate somatic growth responses as a measure for hormone sensitivity. In both treatment studies, ad libitum offspring from both age groups and juvenile IUGR offspring responded to GH treatment However, neonatal IUGR offspring did not exhibit any response to GH treatment.. Analysis of IGF-I gene expression in neonatal offspring showed that GH treatment elevated IGF-I Eb mRNA in ad libitum but not IUGR offspring. These results suggest a possible mechanism for transient GH resistance in that a post-receptor defect in GH action may contribute to the development of temporary postnatal GH resistance as a consequence of IUGR and fetal programming of IGF-I gene expression. In summary, the development of a model of IUGR in the rat using maternal undernutrition throughout gestation has enabled detailed investigation of nutritional regulation of the somatotrophic axis during fetal development and postnatal sequelae. The studies in this thesis have shown that the somatotrophic axis is markedly altered postnatally by nutritional restriction of the dam throughout gestation, leading to prolonged postnatal growth retardation and elevated blood pressure The mechanisms which lead to the induction of such fetal programming and whether these changes may contribute to the development of subsequent adult-onset disease remain to be addressed in future studies.

The developmental origins of health and disease hypothesis suggests that exposure to adverse prenatal environmental influences can determine an individual’s susceptibility to obesity in adult life. However, the specific causal mechanisms which underlie this hypothesis have yet to be identified. Focusing on the potential mechanistic role of the leptin endocrine axis, the main objective of this thesis was to investigate the long term effects of prenatal undernutrition and different levels of postnatal nutrition on leptin sensitivity and the development of diet-induced obesity (DIO) in the Wistar rat. A well established animal model of maternal undernutrition during pregnancy was used to induce prenatal undernutrition in experimental offspring. To investigate the interaction between prenatal nutrition and postnatal diet, and its effects on obesity development, female offspring were placed on three different diets: standard chow, a high fat diet or a calorie restricted diet. The effects of prenatal undernutrition and postnatal diet on leptin sensitivity were investigated, in adult offspring, by measuring the response to 14 days of peripheral leptin treatment. Changes in gene expression in the liver, retroperitoneal adipose tissue and soleus muscle were then characterised by custom microarray and quantitative real-time RT-PCR (QPCR) analysis. Adult female offspring exposed to prenatal undernutrition (UN offspring) were found to exhibit leptin resistance in adulthood, independent of postnatal DIO. This result demonstrates for the first time that exposure to prenatal undernutrition has a long term effect on adult leptin sensitivity. In UN offspring fed on a high-fat diet, leptin resistance significantly accelerated the development of DIO while in contrast, offspring maintained on calorie restriction remained lean. These findings suggest that prenatal nutrition can shape future susceptibility to DIO by altering postnatal leptin sensitivity. An analysis of gene expression suggests that prenatal undernutrition causes the development of peripheral tissue-specific leptin resistance, and may also further enhance an offspring’s susceptibility to DIO by altering the regulation of peripheral tissue lipogenesis, mitochondrial function, glucocorticoid metabolism and insulin sensitivity. In conclusion, these studies identify peripheral leptin resistance as a key mechanism that can influence postnatal susceptibility to DIO in female offspring exposed to prenatal undernutrition. Furthermore, the identification of specific changes in peripheral gene expression highlights four additional metabolic mechanisms which may also facilitate the development of DIO in leptin resistant UN offspring.

Transplacental glucose transport is vital to fetal growth. Although the presence of glucose transporter-1 (GLUT1) and GLUT3 has been demonstrated in mammalian placenta, the factors regulating these genes remain unclear. Therefore, the overall aim of these studies was to clone ovine GLUT1 (oGLUT1) and oGLUT3 cDNAs, and to use these to investigate gene expression during ovine placental development and function. Ovine GLUT1 (~2.2 kb) and oGLUT3 (483 bp) cDNAs were isolated and cloned. Sequence analysis demonstrated that oGLUT1 showed high homology (97 - 99%) with other mammalian species, whereas oGLUT3 did not (84 - 88%). Northern analysis demonstrated that oGLUT1 mRNA abundance increased from d 45 to d 120 of gestation, then decreased towards term (d 145 ± 2), whereas oGLUT3 mRNA abundance increased throughout gestation. Western analysis showed oGLUT1 protein levels increased during late gestation, indicating post-transcriptional regulation of oGLUT1. Localisation experiments revealed spatio-temporal differences in ovine placental GLUT expression. In early gestation (d 45), oGLUT1 protein was restricted to fetal trophoblast cells. By mid gestation oGLUT1 immuno-signal was predominantly localised to maternal villous and endometrial tissue. By late gestation oGLUT1 mRNA was most strongly localised to maternal syncytiotrophoblast and villous tissue, whereas oGLUT3 was predominantly localised to fetal trophoblast cells. Placental oGLUT expression was regulated differently by acute (3 - 8 h) versus long-term (>6 d) alterations in late gestation maternal glucose supply. No evidence was found for regulation of placental oGLUT gene expression by long-term maternal undernutrition, but oGLUT1 and oGLUT3 mRNA and oGLUT1 protein were elevated by short-term (24 - 48 h) maternal hypoglycemia. Acute maternal hyperglycemia transiently increased oGLUT1 and oGLUT3 mRNA abundance, whereas oGLUT1 protein (but not mRNA) levels increased after long-term maternal hyperglycemia. Infusion studies provided no conclusive evidence for regulation of placental oGLUTs by long-term administration of growth hormone (GH) or insulin-like growth factor-1 (IGF-1) to the late gestation fetus. Following acute (4 h) fetal IGF-1 infusion, placental oGLUT3 mRNA abundance was greater in growth restricted (placental embolisation) than in normal fetuses, although the reason for this difference remained equivocal. This thesis describes isolation, cloning and sequence analysis of oGLUT1 and oGLUT3 cDNAs. These studies confirmed the presence of GLUTI and GLUT3 mRNA in ovine placenta, and demonstrated ontogenetic and nutritional regulation of placental oGLUT1 and oGLUT3. In addition, these results indicated that regulation of placental oGLUTs may occur at both transcriptional and post-transcriptional levels.

Whole document restricted, see Access Instructions file below for details of how to access the print copy. / Fetal growth and development is primarily limited by maternal substrate supply. Recent studies also suggest that IGFs and their binding proteins have a significant influence on fetal growth, and that maternal nutritional status is a major factor in the regulation of the IGF/IGFBP axis in fetal plasma. The aim of these studies was a) to develop specific homologous radioimmunoassays (RIAs) for ovine IGFBP-2 and IGFBP-3 for subsequent studies, b) to further define the interactions between maternal nutritional status and circulating IGFs and the IGFBPs in the sheep fetus and c) to characterise aspects of the fetal IGF/IGFBP axis that are temporarily or permanently altered (reprogrammed) in response to limited substrate supply. Such changes might provide mechanistic explanations for epidemiological data linking impaired growth in utero with increased susceptibility to specific diseases in adulthood. IGFBP-2 and IGFBP-3 were isolated from fetal and postnatal sheep serum respectively following 1) cation exchange chromatography to remove endogenous ligand, 2) IGF-2 affinity chromatography and 3) C8 and C18 reverse phase chromatography. N-terminal amino acid sequence analysis revealed strong homology for each peptide with data from several other species. Ovine IGFBP-2 had a 3 amino acid deletion at the N-terminal when compared to human or bovine IGFBP-2, the significance of which is unknown. Specific antisera were raised against both peptides and homologous RIAs were generated. While IGFs did not interfere in either RIA, IGF-1 addition was required in the oIGFBP-3 RIA to overcome a potency difference for the antiserum between purified oIGFBP-3 and plasma IGFBP-3:IGF-1 complex. Both RIAs were validated for analysis of fetal and postnatal sheep plasma. We characterised the roles of insulin and glucose in mediating the effects of substrate supply on plasma IGFs and IGFBPs in the fetal sheep. Pregnant ewes were starved for 72 hours and refed for 48 hours. Fetuses were infused with either glucose or insulin during the final 24hours of maternal starvation. Glucose, insulin, IGF-1 and IGF-2 were reduced by maternal starvation. While glucose infusion increased these parameters to near control values, insulin infusion was only associated with elevations in insulin and IGF-1 concentrations. These data suggest that insulin regulates the fetal plasma levels of IGF-1 but that IGF-2 concentrations were regulated by glucose in an insulin-independent fashion. Fetal plasma IGFBP-1 and -2 were increased, and IGFBP-3, IGFBP-4 and the circulating IGF-2/mannose-6-phosphate receptor (IGF-2/M6PR) were decreased, by maternal starvation. IGFBP-1 and IGFBP-4 levels were restored to normal by glucose and insulin infusions while the IGF-2/M6PR was increased by glucose infusion only (not measured in insulin-infused fetuses). These results are consistent with data regarding firstly the insulin dependency of IGFBP-1 and its role as a glucose counter-regulatory peptide, and secondly the role of insulin in determining IGFBP-4 and IGF-2/M6PR distribution between plasma and tissues. Fetal plasma IGFBP-2 and -3 levels were unaffected by either infusion suggesting that they are regulated by factors other than acute changes in plasma glucose or insulin. We have subsequently examined the hypothesis that exposure to undernutrition around the time of conception would result in a reduced growth rate and reprogramming of the plasma IGF/IGFBP axis in the late gestation fetus. Pregnant ewes were either well fed or undernourished from 60 days prior to mating to d30 of gestation and then subjected to a second period of undernutrition between d105 and d115 of gestation. Periconceptual undernutrition was associated in the late gestation fetus with reduced growth rate and a reduction in the circulating concentrations of IGFBP-1 and IGFBP-3. Furthermore the response of plasma IGF-1, IGFBP-1 and IGFBP-3 in fetuses of the periconceptual undernutrition group to severe maternal undernutrition in late gestation was significantly greater than that measured in fetuses from mothers well fed at the time of conception. The hypothesis that a severe nutritional insult only during late gestation would alter the postnatal regulation of plasma IGF-1 and IGFBP-3 in the lamb was also addressed. While birth weight was reduced in fetuses of mothers undernourished for 20 days, no differential effects of undernutrition in utero on the pre-pubertal ontogeny of IGF-1 and IGFBP-3 or on their response to a GH bolus could be found between the treatment groups. In summary, these studies indicate that IGFs and IGFBPs play an important role in mediating the effects of fetal substrate supply on fetal growth. Reprogramming of the responses by the plasma IGF/IGFBP axis to undernutrition in the late gestation sheep fetus could be induced by a period of periconceptual undernutrition. However, the lack of reprogramming effects on IGF-1 and IGFBP-3 in postnatal plasma following a severe nutritional insult only in late gestation suggests either that reprogramming of the IGF/IGFBP axis is dependent on exposure to undernutrition at earlier timepoints in gestation or that reprogramming of the fetal IGF/IGFBP axis does not persist postnatally.

The experiments described in this thesis investigated the effects of low doses of insulin-like growth factor (IGF)-I on fetal growth and metabolism in normally growing and growth-restricted (IUGR) late-gestation ovine fetuses. Intra-amniotic and intravenous routes of administration were studied. The aim of the first experiment was to investigate the effects of daily intra-amniotic injections of IGF-I for l0 days to fetuses with IUGR induced by placental embolisation. Embolisation produced asymmetrical IUGR with moderately severe effects on the gut. Gut weight, gut wall thickness and crypt mitoses were reduced. Villus enterocytes in the ileum contained numerous vacuoles, suggesting either functional adaptation or delayed maturation. IGF-I treatment increased amniotic fluid IGF-I concentrations 5-fold, but decreased plasma concentrations. The effects of embolisation on the gut were reversed, except for the enterocyte vacuolation in the ileum which was more pronounced with IGF-I treatment. Fetal gut uptake of glutamine from the circulation was reduced in IGF-I treated animals, but uptake from amniotic fluid may have increased. Fetal liver and spleen size were reduced. The distribution of placentome types was also altered with IGF-I treatment. The aim of the second experiment was to investigate the clearance of 125I-IGF-I from amniotic fluid. The half-life of 125I-IGF-I in amniotic fluid was 24 hours. Significant amounts of 125I-IGF-I remained unbound for up to 144 hours in both amniotic fluid and plasma. The predominant binding protein in amniotic fluid was IGFBP-3, and the proportion of 125I-IGF-I that was bound in amniotic fluid was strongly correlated with relative levels of IGFBP-3. Uptake of intact 125I-IGF-I across the fetal gut was clearly demonstrated, and continued for at least 36 hours following injection. A preliminary pharmacokinetic model of intra-amniotic dosing with IGF-I is presented. The aim of the final experiment was to investigate the effects of a chronic intravenous infusion of a low dose of IGF-I to normally growing late-gestation fetuses. There were no effects of IGF-I treatment on fetal growth or metabolism. Five fetuses were found to be IUGR at post mortem due to the effects of facial eczema in the ewe. A post hoc analysis of the effects of intravenous IGF-I in IUGR fetuses was therefore performed. IGF-I treatment significantly increased growth rate of IUGR fetuses and increased fetal blood aminonitrogen levels. Mean placentome weight was increased and the distribution of placentome types was altered. IGF-I treatment of IUGR fetuses reduced fetal lactate production. There were no differences in measures of fetal body or organ size at post mortem. The data from these investigations into the experimental manipulation of fetal growth provide clear evidence of the reversal of some of the effects of established IUGR, demonstrate uptake of intact and free IGF-I across the fetal gut, and, for the first time, suggest that IGF-I may increase the rate of fetal growth in IUGR. Amniotic fluid is the most readily accessible fetal compartment. The experiments described in this thesis suggest that fetal supplementation with IGF-I via the amniotic route may be a feasible, efficacious and clinically applicable therapeutic stratagem for the IUGR fetus. / Whole document restricted, but available by request, use the feedback form to request access.