This poststructuralist qualitative study explored the discourses constructing women’s choice for a caesarean section in the absence of clinical indications, in the talk and texts of women, midwives, an obstetrician, professional journals and the media publications. The study affirms inscriptions surrounding choice in childbirth are shaped discursively through a multiplicity of discourses underpinned by social and institutional practices. With advances in technology, childbearing women have a greater variety of options from which to choose. Controversial, is the option of a caesarean section, regardless of clinical need. The issue is depicted in both professional and popular discourse as contentious, complex and contradictory. Its momentum into the 21st century, as a new object of obstetric discourse, has been played out on a number of platforms. In this thesis I draw from the theoretical ideas of French philosopher Michel Foucault, to examine this complex debate. I argue there is a volatile moment in the history of childbirth in which an explosion of discourses have sculptured choice for a caesarean, in the absence of clinical indications, out of a repartee of autonomy, convenience, desire, fear and risk. In this precarious moment, new meanings joust with the old on a shifting terrain awash with rhetoric that co-opts, competes, and contradicts to bring about a caché of mutable ‘truths’. Whether caesarean, as an optional extra, can be explained in terms of a libertarian imperative, an embodiment of lifestyle, the satiation of desire, the attenuation of fear or the avoidance of risk, the democratisation of this choice has exposed a pathologising paradox, whereupon the normal emerges as the abnormal, and the abnormal emerges as the normal. The deconstruction of choice through a poststructuralist lens has enabled insight into how contradiction and contest befall the ‘order of things ’ and in so doing, provides new openings for contemplating the discursive positioning of women through the competing discourses of childbirth.

Testicular function is stimulated from the pituitary gland by the gonadotrophic hormones FSH and LH. The testis in turn regulates gonadotrophin secretion by negative feedback. The agents of feedback are steroids, and in addition, probably a protein hormone, which has been given the name inhibin. A method capable of detecting inhibin-like activity in bull seminal plasma (bSP) was developed. Administration of bSP at the time of castration to five-week old male rats inhibited the post-castration rise of serum concentrations of FSH and LH otherwise seen 24 h later. The degree of inhibition depended on the dose; 0.5 ml bSP or the equivalent amount of bSP-extract always suppressed FSH and LH to levels typical of intact rats. The rats' sensitivity to 0.2 ml bSP varied with the time of year, possibly reflecting seasonal changes in the onset of puberty. The time-course of action of bSP-extract was studied in intact rats. Serum FSH was suppressed 12, 24 and 48 h after a single injection, but not at 3 or 6 h. LH was suppressed at 6, 12, 24 and possibly 48 h.

In the first trimester of human pregnancy specialised placental cells, termed cytotrophoblasts differentiate into extravillous trophoblasts (EVTs), which grow out from the placenta and invade into the maternal decidua, acting to physically attach the placenta to the decidua, and adapt the uterine spiral arteries to support pregnancy. A proportion of these EVTs also temporarily occlude the spiral arteries for approximately the first 10 weeks of gestation, preventing maternal blood flow to the placenta and creating a low oxygen environment in which placental and fetal development occur. The processes of trophoblast outgrowth and invasion, and the establishment of a low oxygen environment, are essential for the success of pregnancy, and inadequate trophoblast invasion into the uterus has been associated with recurrent miscarriage, pre-eclampsia and fetal growth restriction. However, the exact mechanisms that control the differentiation of cytotrophoblasts down the extravillous trophoblast lineage are poorly understood. Since the extent of this invasive process is unique to human implantation, animal models are of limited value in studying trophoblast invasion. Existing in vitro models have major limitations in that many are very difficult to quantify while others many not study the correct trophoblast population. The research in this thesis has focussed on the development of novel models by which to study cytotrophoblast differentiation, and the use of these models to further understand cytotrophoblast differentiation down the EVT lineage, and the regulation of EVT outgrowth by oxygen. Methods Outgrowth from first trimester villous explants was characterized using immunohistochemistry. Explant viability was investigated using dual staining with chloromethylfluorescin diacetate (CMFDA) and ethidium bromide, by examining DNA laddering, and by immunostaining sectioned explants over 96 hours of culture. The extended viability of cytotrophoblasts in multilayered cell islands in villous tips was exploited to isolate these cells using sequential trypsin digests of cultured villous explants. The trophoblast population obtained were characterized by immunohistochemistry. Finally, villous explants were cultured in either 1.5% or 8% oxygen and the frequency and area of outgrowths was quantified in order to determine the effect of gestation and oxygen on EVT outgrowth. Results Approximately 1/4 of explants cultured in 20% oxygen produced EVT outgrowth. Outgrowth formation and expansion resulted from proliferation of cells in the tips of anchoring villi, and EVTs within the outgrowth did not proliferate. The percentage of explants producing outgrowth declined as gestation increased from 8 to 12 weeks. Dual staining with CMFDA and ethidium bromide revealed degeneration of the syncytiotrophoblast by non-apoptotic mechanisms within 4 hours of culture, but this syncytiotrophoblast layer was able to be regenerated. The majority of cytotrophoblasts died within one week of culture, but despite this explants were able to produce EVT outgrowth for up to 3 weeks due to the extended survival of a specific set of cytotrophoblasts located in cell islands in the tips of anchoring villi. These surviving cells were able to differentiate into EVTs, but not regenerate the surrounding syncytiotrophoblast in the villus tip. Trypsinization of first trimester villi after extended explant culture resulted in the isolation of a viable population of ‘putative EVT progenitors’ that did not syncytialise in culture, but were able to proliferate. 20% of these cells differentiated down the EVT lineage within 96 hours of culture. The putative EVT progenitors expressed markers previously localised to cytotrophoblasts in cell islands of anchoring villi, including αvβ6 integrin and FGFR-2. The addition of exogenous FGF-4 did not affect the differentiation of these cells into EVTs, nor did FGF-4 alter the frequency of EVT outgrowth from explants. Culture in 1.5% oxygen significantly reduced the frequency and area of outgrowths in comparison to 8% oxygen. HLA-G and α1 integrin were both expressed throughout outgrowths with no difference in expression of these proteins between oxygen concentrations. Gestation influenced the response of explants to oxygen, with a significant differential response to oxygen concentration in placentae under 11 weeks of gestation but no differential response in placentae of 11 or 12 weeks. Conclusions In the first trimester, oxygen and gestational age regulate extravillous trophoblast outgrowth in both an independent and interdependent manner. The cytotrophoblast population in the first trimester does not consist of one homogenous bipotent population. Rather there are at least two separate populations: 1) EVT progenitors that exist in the tips of potential anchoring villi that are likely to be committed to EVT differentiation and 2) monolayer villous cytotrophoblasts which are likely to be committed to syncytiotrophoblast differentiation. The second population is easily isolated by traditional enzymatic digestion methods whereas the first much smaller population can be isolated by exploiting their prolonged survival in explant culture.

Human mammary tissue was incubated in vitro with [7-3H] dehydroepiandrosterone sulphate (DHA-sulphate) and in agreement with other investigators this steroid conjugate was metabolized to DHA and other steroid products. Sulphatase activity was greater in the malignant than the non-malignant tissues and was found to be a function of the tissue cellularity. One of the major products, a "polar steroid" necessitated identification. The "polar steroid" was identified principally as 7a-hydroxy DHA by chemical modification techniques and co-crystallization of the purified steroid metabolite with carrier 7a-hydroxy DHA. This carrier required synthesis and characterization.

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. / Zinc is an important trace metal in human biology. It plays an active role in enzymic reactions, or simply serves in a structural capacity on a number of cytoplasmic and nuclear proteins. Zinc modulates receptor responses to various excitatory and inhibitory neurotransmitters. It has biphasic effects on several enzymes critical for cell survival and the induction of apoptotic cell death. At a particular concentration threshold, it is cytotoxic both in vitro and in vivo. Recent studies have indicated that zinc may be involved in several neuropathological conditions such as Alzheimer's disease, epilepsy, traumatic head injury and cerebral stroke. This investigation focused on the role of zinc in the context of an Alzheimer's disease paradigm. Rat primary cortical neurons were exposed to freshly-prepared (non-aged) or aggregated (aged) Aβ1-42 protein (20 µM; a highly toxic Aβ species), in the presence or absence of equimolar concentrations of zinc chloride (ZnCl2), or copper chloride (CuCl2).Zinc significantly attenuated, while copper potentiated the neurotoxic effects of non-aged Aβ1-42 after 48 h chronic exposure. Similarly, zinc, but not copper, reduced neuronal death 48 h following exposure to the aged peptide. A metal chelator, DTPA, also showed a protective, but limited effect (only observable at 24 h post-treatment) against the neurotoxicity of aged peptide. At the concentration tested, zinc alone had no effect on neuronal survival, although copper was found to be slightly neurotoxic after 48 h incubation. As hydrogen peroxide (H2O2) production by Aβ has been reported to mediate its cytotoxicity, an in vitro test system was used to identify if zinc affected this process. It was found that co-incubation of zinc (10 µM) with Aβ1-42 peptide alone (10 µM) or with Aβ1-42 and copper (1 µM) showed a significant decrease of Aβ-mediated H2O2 formation using TCEP assay in vitro. This finding suggested that the neuroprotective effect of zinc may not only be due to its capacity to hinder Aβ's redox activity, but also zinc's ability to preclude Aβ-mediated H2O2 generation. A mechanism for these effects of zinc is yet to be determined. As oxidative/nitrosative stress has been widely reported to occur in AD brain, and since zinc metabolism is believed to be dysfunctional in AD brain, the current study also set out to elucidate if cerebral zinc metabolism may be affected by nitrosating agents in vivo. Three unrelated nitric oxide-generating compounds were administered into rat hippocampus. Using Timm's and TSQ stain, a histochemical and a fluorescent staining for zinc, respectively, it was observed that sodium nitroprusside (≥2 nmol) and spermine-nitric oxide complex (≤200 nmol), but not the peroxynitrite-producing agent 3-morpholinosydnonimine (≤200 nmol), caused perikaryal zinc accumulation among certain neurons in the hippocampus. Both membrane impermeable and permeable metal chelators, EDTA and TPEN, respectively, blocked perikaryal zinc staining of hippocampal neurons. Data obtained from EDTA treatment suggested that the source of perikaryal zinc staining was mostly extracellular. Previous reports have shown that metal chelating agents have the capacity to protect neurons and minimize damage from brain insults. The preceding studies showed that chelators minimized perikaryal zinc acccumulation, precluded Aβ's redox activity, and partly reduced Aβ neurotoxicity. Thus, to further assess the possible beneficial effect of these compounds, DTPA, BP, or BC (25 µM) was incubated with a mixture of Aβ1-42 (2 µM) and Aβ1-40 (20 µM). DTPA abolished, while BP delayed the Aβ1-42-mediated Aβ1-40 "seeding" process. This result suggested that contaminating trace metals have an obligatory role in the nucleation-dependent Aβ fibrillogenesis, which is believed to be linked to Aβ neurotoxicity, and AD neuropathology. These results imply that zinc has a biphasic role in AD etiology and disease progression, and that the use of metal chelators to buffer pathologically excessive zinc and other metals, particularly the redox active copper and iron, may have a potential therapeutic value against AD symptomatology.

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 nexus or linkages between loneliness, stigma, health complaints, and primary medical care in older New Zealanders was explored from a social constructionist perspective. The intent of the studies was the support and explanation of the underlying arguments of the thesis. For this age group loneliness is a clinical condition which merits greater recognition, diagnosis, and treatment from general practitioners than it presently receives. As a society we silence and stigmatise loneliness in our senior citizens making it likely that they will present indirectly to their doctors when experiencing severe effects of the condition. This behaviour will increase their risk of inappropriate medical intervention at possible cost to themselves and to society. A cross sectional, randomly selected survey of 300 New Zealanders over 60 years old, aimed to establish the patterns of loneliness in the sample using quantitative analysis. The second qualitative study used the methodology of discourse analysis to identify the themes concerning loneliness and medical care in the accounts of older adults, and how these were used. Fourteen people, deemed by their doctors to be lonely and to need frequent medical care, were interviewed in order to further knowledge of the dynamics of loneliness and the medical encounter. Fifteen percent of the sample of 300 had moderate to severe loneliness scores. The sociodemographic indicators of loneliness were extremely easy for a practitioner to recognize. Less than 2% of the total of self reported doctor visits were explicitly for loneliness. According to Barsky's (1981) model, the most likely pathways to the doctor were through symptom amplification and lowered self ratings of health, with a less likely pathway through focusing on and worrying about symptoms, leading to perceived need for medical care. The predictive variances in regressions of loneliness on all health outcomes, except for self reported visiting of more than one doctor for symptoms, were lower for chronic than for situational loneliness. The most important conclusions from the second study were the identification of three rhetorical strategies or "etcetera clauses" which provided a social prescription for the indirect presentation of loneliness by older people. Loneliness may be discussed with the doctor; if it affects your physical health; if you are consulting for another reason; and if the doctor picks it up. Also, the individual doctor defines loneliness as a worthy, or non-worthy, condition for consultation.

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.

Observations that IGF is produced and acts locally in multiple tissues raise important questions about the biological significance of the major pool of IGF present in the circulation. Does it represent a pool of endocrine IGF en route to the tissues or conversely growth factor produced in excess of autocrine/paracrine requirements undergoing elimination? The primary objective of this thesis was to examine the kinetics and distribution of circulating IGF in sheep with a view to determining tissue destinations and thereby potential functions of the blood borne hormone. The IGFBP play a central role in facilitating IGF action. Characterization studies of the IGFBP and an examination of their physiology and potential involvement in IGF transport are also important parts of this thesis. Such studies are necessary because potential therapeutic uses of IGF will depend on systemic administration and endocrine action. Early work involved structural/functional characterization of a batch of recombinant methionyl insulin-like growth factor-I (N-Met IGF-I) designated for this project. The peptide was heterogenous on reversed phase chromatography eluting as two major peaks of approximate abundance 1:2. These each had the amino acid constitution expected of N-Met IGF-I and were carefully characterized in a range of binding and biological assays. Whereas the early eluting peptide demonstrated much reduced activity in each assay system, the second peak proved equipotent to a highly purified ovine plasma IGF-I preparation and was chosen for the investigative work of this thesis. The early eluting peptide may represent a variant with mismatched disulphides. Initial characterization of IGF binding activity in ovine tissue fluids was performed by competitive IGF tracer binding techniques together with size exclusion chromatography (SEC) and IGF-I affinity chromatography. Binding proteins (BP) of >200, 150 and 40-50 kDa were revealed in these studies and shown to be widely distributed in body fluids. Thus the >200 kDa binding protein, which is IGF-II specific, was identified in adult sheep plasma, colostrum, follicular fluid and fetal sheep plasma, and may be the ovine equivalent of the soluble type 2 IGF receptor. A 150 kDa binding protein complex, of mixed specificity for IGF-I and II, was also identified. In addition to vascular fluids, the 150 kDa complex was identified in mammay lymph, follicular fluid and, as a minor component, in vitreous humor. Binding proteins of 40-50 kDa were revealed in every fluid tested and multiple variants identified with distinct specificities for the IGF peptides. The BP 'make-up' of fluids and of 150 kDa and 40-50 kDa pools isolated by preliminary SEC was latter examined by IGF ligand blot analysis. Analysis of plasma 150 kDa pools revealed only the characteristic doublet of IGFBP-3 at 40-43 kDa, whereas the 40-50 kDa pool was heterogeneous containing IGFBP-3 together with smaller bands of 35, 30 and 23 kDa which may be the ovine equivalents of IGFBP-2, BP-4 and possibly BP-1. In support of the tracer binding data, IGFBP-3 was also identified in mammary lymph as were the smaller species. In an extension of the in vitro IGF tracer binding/SEC approach, kinetics of IGF equilibration with plasma binding sites was examined. Binding was found to be time and temperature dependent, reversible, dose responsive and relatively specific for the IGF peptides. Observations of special interest include a biphasic pattern of IGF-I equilibration with plasma, consistent with formation of the ternary 150 kDa complex of IGFBP-3, IGF and ALS, and evidence of relatively slow dissociation of IGF/BP complexes, suggesting that if release of IGF is required for full expression of IGF bioactivity in vivo, then specific processes may be involved. Avidity of isolated IGFBP complexes for Con A and heparin affinity adsorbents was also examined. The data indicate that the IGFBP belong to a relatively select group of proteins with high affinity for the glycosaminoglycan heparin suggesting roles for these proteins at the level of the capillary endothelium and/or extra-cellular matrix. Metabolic clearance of IGF-I and II was examined following intravenous (iv) bolus injection of the growth factors as radioiodinated tracer preparations. Tracer administration was followed by a rapid initial phase of clearance associated with tracer mixing in the vascular pool followed by intermediate and longer phases which appear to be direct consequences of interaction with and between the BP and to some extent accumulation of tracer degradation products in the circulation. Metabolic clearance of tracer complexed to the major molecular weight pools of BP was examined following SEC of sequential plasma samples. Average half-lives for IGF-I and II complexed to the 150 KDa and 40-50 kDa pools of carrier protein were established (150 kDa; 545±25 min, 325±30 min; 40-50 kDa, 34±2 min, 9.6±1.8 min, (mean±S.E.M., IGF-I and II respectively)) and compared to free IGF-I (t1/2 <5 min). Rapid clearance of free compared to bound IGF illustrates the central role of the IGFBP in maintaining IGF in the circulation and controlling tissue distribution. Whereas binding of IGF-II to different BP at 40-50 kDa (eg. IGFBP-2) may explain its shorter half-life compared with IGF-I, evidence suggests that IGF-I and II bind to the same carrier at 150 kDa. The observed difference in half-life of the 150 kDa complex is therefore suggestive of different metabolic handling of the BP depending on which of the IGFs is bound. The more rapid clearance of IGF-II complexed to the 150 kDa and 40-50 kDa carriers compared to IGf-I contributed to a more rapid clearance overall and is reflected in calculated metabolic clearance rates for IGF-I and II (IGF-I, 3.9 ml/min; IGF-II, 7.8 ml/min). Considering plasma IGF-II is significantly higher than IGF-I in post-natal sheep, a substantially greater secretion rate for IGF-II would be required to maintain plasma IGF-II in the face of the greater clearance rate. The secretion rate for IGF-II was estimated at ~ 1.6 nmol/min in the current study, some 8-fold greater than IGF-I. Clearance of IGF-I from plasma was associated with the appearance of radioactivity in lymph. Chromatography indicated that tracer in lymph was not degraded but retained its BP activity eluting on SEC complexed to high molecular weight BP. The data illustrate that blood borne IGF is distributed into the extra vascular space and may therefore be available to the tissues. This contention is supported by observations that relatively little radioactivity (<20% in the course of these experiments) was cleared from plasma into urine suggesting that plasma IGF is not principally an elimination form. Similarly no other significant sites of elimination were identified. Questions of how physiological control of the BP may influence tissue distribution of IGF were investigated in the next major experimental section of this thesis. In the first study the influence of nutritional manipulation and GH treatment of growing lambs on the molecular distribution of IGF immunoreactivity in plasma was examined using a new IGF-I RIA in conjunction with SEC and saturation analysis for the estimation of the BP. Total plasma IGF-I was found to increase with nutritional intake (P<0.01) and with GH treatment (0.25 mg/kg body weight/d; P<0.001) but only on the higher intakes. Molecular size fractionation revealed IGF-I immunoreactivity in 150 kDa and 40-50 kDa binding fractions. 150 kDa bound IGF-I was increased on the higher plane of nutrition(P<0.05) and by GH treatment (P<0.001) but again, only at higher levels of nutrition. By contrast no change in 40-50 kDa bound IGF-I was observed with treatment. Unbound IGF-I was also identified in sheep plasma (2-5% of total) but demonstrated only slight changes in relation to treatment. Saturation analysis was an analytical approach chosen to estimate total binding capacity (TBC) and relative saturation of the binding protein pools. Evidence suggests that in ovine plasma constituents of the 150 kDa complex are available in excess of endogenous IGF (P<0.001). Relative saturation of this species did not change with treatment despite the observed differences in 150 kDa bound IGF-I. The data suggest that components of the 150 kDa complex were themselves responsive to treatment. By contrast large differences in saturation of the 40-50 kDa species were observed (P<0.001) despite little treatment dependent change in bound IGF-I. Binding capacity of the 40-50 kDa fraction was elevated at low levels of nutrition and suppressed on the higher feed intake resulting in near saturation. The data indicate complex regulation of the IGFBP in sheep. IGF-I, elevated in response to higher nutritional intake and by CH treatment was mostly distributed into the 150 kDa complex; paradoxically the species which most effectively maintains IGF in the circulation. Thus in conditions presumably conducive with growth related processes (high GH, high nutrition) access of circulating IGF to the tissues is apparently most restricted. This evidence is difficult to reconcile with the view that 150kDa bound IGF represents a pool of endocrine IGF en route to tissue sites of action. Galactopoietic effects of GH in lactating ruminants appear to be exerted in the absence of a mammary GH receptor and are associated with increased plasma and mammary IGF-I content. Thus it has been proposed that blood borne IGF, acting in the classical endocrine fashion may be the mediator of GHs lactogenic effects. Consequently the lactating sheep surgically prepared by the catheterization of efferent mammary lymph may be a useful model for examining questions of IGF/BP physiology. In a further study, plasma and efferent mammary lymph concentrations of IGF-I were determined in lactating ewes before and after treatment with GH (10 mg/d) for 3 days. Analysis of paired plasma/lymph samples revealed that the capillary endothelium constitutes a barrier to the passage of macro molecules which reduces the concentration of IGF in lymph to ~ 35% plasma. A key observation from the current study was the GH dependence of mammary lymph IGF-I. Thus, GH was found to increase mammary lymph IGF-I concentrations by a proportionately greater amount than the increase in plasma IGF-I (P<0.01). The increase in lymph IGF-I resulted from an increase in the concentration of IGF associated with both high molecular weight (150 kDa) and low molecular weight (40-50 kDa) binding fractions. However, the data indicate a proportionately greater increase in 40-50 kDa bound IGF-I in lymph compared with plasma suggesting that treatment either induces a selective redistribution of plasma 40-50 kDa IGF and BP into the mammary gland or, alternatively, treatment increases intra-mammary production of these factors. Ligand blot analysis of mammary lymph revealed IGFBP-3 and -2 as the major constituents of this fluid. IGFBP-2 declined in lymph with GH treatment whereas IGFBP-3 appeared to increase. Additionally, saturation analysis indicated that a substantial proportion of lymph IGFBP-3 was present in the 'free', uncomplexed form. Consequently observations that the total binding capacity (TBC) of the lymph 40-50 kDa fraction increased with treatment, would appear to result from an increase in IGFBP-3. Total binding capacity of the lymph 40-50 kDa binding fraction was found to increase by a proportionately greater amount that its plasma equivalent. Thus, if the lactogenic effects of GH are mediated by IGF distributed from blood into the mammary gland, the mechanism by which it is transferred would appear to involve BP of the 40-50 kDa pool and in particular IGFBP-3. In the final experimental section of this thesis a novel system was employed to examine tissue distribution and destinations of blood borne IGF-I. This involved intravenous infusion of the N-Met analog of IGF-I together with specific immunologic detection. For this an IGF antibody was employed which recognises the recombinant N-Met variant but demonstrates minimal cross-reactivity with any of a range of other IGF peptides including ovine plasma IGF-I and recombinant authentic sequence IGF-I. This antibody can therefore recognise the N-Met variant against a background of authentic endogenous IGF-I and was usefully applied to examining tissue destinations of the N-Met variant following iv infusion. Plasma N-Met IGF-I rose to plateau concentrations of ~150 ng/ml during iv infusion (Infusion rate, 8 µg/kg/h). Analysis revealed the N-Met variant was distributed on plasma BP in much the same proportion as endogenous IGF. N-Met IGF-I immunoreactivity was identified in mammary lymph providing further evidence supporting the contention that blood borne IGF is distributed outside the vascular space. At the end of the infusion N-Met IGF-I was identified in all tissues examined contributing between 35% (in kidney) and 62% (spleen) to total IGF. Major differences in morphological distribution of blood derived (N-Met IGF) were revealed by autoradiography in post infusion tissue slices. Thus the N-Met IGF was found to contribute relatively little to total localizable IGF-I immunoreactivity in connective tissue elements of the samples examined (muscle and mammary) but, by contrast, blood derived (N-Met) IGF-I constituted ~85% of total IGF immunoreactivity in other tissues. In particular, these include the metabolically active regions of muscle and mammary (fibre and epithelium respectively). The evidence suggests therefore that fibre and epithelium may be targets for blood derived ‘endocrine' IGF. Differences in the abundance of blood derived IGF between tissues may relate to the accessibility (vascularization or capillary permeability) of specific tissues or, alternatively, to local rates of production and turnover of IGF in tissues. Thus the contribution of blood derived IGF to total localizable IGF may be expected to be less in tissues which actively synthesize IGF such as those in which autocrine/paracrine modes of IGF action are operative. Examples from the current study would be connective tissues of muscle and mammary. Conversely blood derived IGF would be expected to represent a greater proportion of total IGF in tissue targets for endocrine IGF. Support for the current data was obtained from IGF-I mRNA in situ hybridization studies performed on human fetal tissue (437). Stromal elements of muscle tissue (perimysium, epimysium) were identified as active sites of transcription as opposed to muscle fibre where message could not be detected. Thus the evidence suggests that the N-Met infusion model is a useful technique for delineating tissue targets for circulating (endocrine) IGF. It is now widely accepted that the primary actions of IGF on growth and development occur via autocrine/paracrine mechanisms close to its site of production. Nonetheless such arguments do not exclude the possibility of classical endocrine roles for the major pool of IGF present in the circulation. The primary thrust of this thesis has been to examine kinetics and tissue distribution of this pool of IGF. The data confirm the availability of blood borne IGF to extra vascular tissues and appear to indicate that it is distributed into the tissues on a selective basis and under physiological control. It may, therefore, be available to selected tissues to fill specific endocrine functions. / Whole document restricted, but available by request, use the feedback form to request access.