Foetal and post-natal exposure of sheep to sewage sludge chemicals disrupts sperm production in adulthood in a subset of animals.

Exposure to ubiquitous, environmental chemicals (ECs) has been hypothesized as a cause for declining male reproductive health. Understanding the long-term effects of EC exposure on reproductive health in humans requires animal models and exposure to 'real life', environmentally relevant, mixtures during development, a life stage of particular sensitivity to ECs. The aim of this study was to evaluate the effects of in utero and post-natal exposure to environmentally relevant levels of ECs, via sewage sludge application to pasture, on the adult male sheep testis. Hormones, liver concentrations of candidate ECs and Sertoli and germ cell numbers in testes of adult rams that were exposed to ECs in sewage sludge in utero, and until weaning via maternal exposure, and post-weaning via grazing pastures fertilized with sewage sludge, were quantified. Evaluated as a single group, exposure to sludge ECs was without significant effect on most parameters. However, a more detailed study revealed that 5 of 12 sludge-exposed rams exhibited major spermatogenic abnormalities. These consisted of major reductions in germ cell numbers per testis or per Sertoli cell and more Sertoli cell-only tubules, when compared with controls, which did not show any such changes. The sludge-related spermatogenic changes in the five affected animals were significantly different from controls (p < 0.001); Sertoli cell number was unaffected. Hormone profiles and liver candidate EC concentrations were not measurably affected by exposure. We conclude that developmental exposure of male sheep to real-world mixtures of ECs can result in major reduction in germ cell numbers, indicative of impaired sperm production, in a proportion of exposed males. The individual-specific effects are presumed to reflect EC effects on a heterogeneous population in which some individuals may be more susceptible to adverse EC effects. Such effects of EC exposure in humans could have adverse consequences for sperm counts and fertility in some exposed males.

Effects of exposure to environmental chemicals during pregnancy on the development of the male and female reproductive axes.

There is a large body of literature describing effects of environmental chemicals (ECs), many of them anthropogenic with endocrine-disrupting properties, on development in rodent laboratory species, some of which lead to impaired reproduction and adverse health. This literature joins extensive human epidemiological data and opportunistic wildlife findings on health effects of ECs. In contrast, the effect of endocrine disruption on foetal development and reproductive performance in domestic species is less extensively documented. This applies both to domestic farm and to companion species even though the former is critical to food production and the latter share our homes and many aspects of the modern developed human lifestyle. In domestic species, the nature of chemicals exposure in utero and their consequences for animal health and production are poorly understood. A complication in our understanding is that the pace of development, ontogeny and efficiency of foetal and maternal hepatic and placental activity differs between domestic species. In many ways, this reflects the difficulties in understanding human exposure and consequences of that exposure for the foetus and subsequent adult from epidemiological and largely rodent-based data. It is important that domestic species are included in research into endocrine disruption because of their (i) wide variety of exposure to such chemicals, (ii) greater similarity of many developmental processes to the human, (iii) economic importance and (iv) close similarities to developed world human lifestyle in companion species.

Influence of prenatal nutrition and obesity on tissue specific fat mass and obesity-associated (FTO) gene expression.

The recent discovery of an association between body composition, energy intake and the fat mass and obesity-associated (FTO) gene represents a promising new therapeutic target in obesity prevention. In a well, pre-established large animal model, we investigated the regulation of FTO gene expression under conditions either leading to obesity or increased risk of obesity related disorders: i) a sedentary 'Western' lifestyle and ii) prenatal exposure to nutrient restriction. Pregnant sheep were either fed to fully meet their nutritional requirements throughout gestation or 50% of this amount from early-to-mid gestation. Following weaning, offspring were either made obese through exposure to a sedentary obesogenic environment or remained lean. A significant positive relationship between placental FTO gene expression and fetal weight was found at 110 days gestation. In both the newborn and adult offspring, the hypothalamus was the major site of FTO gene expression. Hypothalamic FTO gene expression was upregulated by obesity and was further increased by prenatal nutrient restriction. Importantly, we found a strong negative relationship between the hypothalamic FTO gene expression and food intake in lean animals only that may imply FTO as a novel controller of energy intake. In contrast, FTO gene expression in the heart was downregulated in obese offspring born to nutrient restricted mothers. In addition, FTO gene expression was unaffected by obesity or prenatal diet in insulin-dependent tissues, where it changed with age possibly reflecting adaptations in cellular energetic activity. These findings extend information gained from human epidemiology and provide new insights into the regulation of in vivo energy metabolism to prevent obesity.

Maternal and fetal tissue accumulation of selected endocrine disrupting compounds (EDCs) following exposure to sewage sludge-treated pastures before or after conception.

Liver concentrations of selected pollutant classes were determined in groups of sheep fetuses and their dams, at 55 (Experiment 1) and 110 (Experiment 2) days of gestation (term = 145 d) following exposure, throughout their breeding lives and after mating, to pasture treated with either inorganic fertiliser (control, CC) or with sewage sludge (treated, TT). In a unique study designed to separate the respective contributions of environmental sources and mobilised tissue to the available EDC burden, in additional groups of animals, pollutant burdens at 110 days gestation were assessed following exposure to the respective treatments, either throughout their breeding lives until mating, but not thereafter (TC), or only between mating and slaughter (CT) (Experiment 3). With very few exceptions, maternal and fetal liver concentrations of diethylhexyl phthalate (DEHP) and selected polychlorinated biphenyls (PCBs), and polybrominated diphenyl ethers (PBDE) and polycyclic aromatic hydrocarbons (PAHs) were not significantly affected by sludge exposure in any group. In some cases, maternal and fetal tissue EDC concentrations were different but the differences were not consistent, and maternal and fetal concentrations of none of the classes of chemical were significantly correlated. It was not possible to identify a single chemical, or class of chemical, that may be responsible for previously observed physiological effects of exposure to sludge-treated pastures. It is concluded that exposure of sheep to pastures fertilised with sewage sludge was not associated with increased liver concentrations of EDCs, irrespective of the stage of development at which they were measured and of maternal tissue mobilisation and EDC release during gestation. Thus, retrospective measurements of EDC tissue burdens could not be used to accurately assess earlier fetal EDC insults.

The differential effects of the timing of maternal nutrient restriction in the ovine placenta on glucocorticoid sensitivity, uncoupling protein 2, peroxisome proliferator-activated receptor-gamma and cell proliferation.

Nutrient restriction (NR) during critical windows of pregnancy has differential effects on placento-fetal growth and development. Our study, therefore, investigated developmental and metabolic adaptations within the ovine placenta following NR at different critical windows during the first 110 days of gestation (term=147 days). Thus, the effects of NR on cell proliferation, glucocorticoid sensitivity, IGF1 and 2 receptor, peroxisome proliferator-activated receptor gamma (PPARG), and uncoupling protein (UCP)2 gene expression in the placenta were examined. Singleton bearing sheep (n=4-8 per group) were fed either 100% of their total metabolizable energy requirements throughout the study or 50% of this amount between 0-30, 31-65, 66-110, and 0-110 days gestation. A significant reduction in cell proliferation and increased gene expression for the glucocorticoid and IGF2 receptors, PPARG, and UCP2 were detected in placentae sampled from mothers who were nutrient restricted between days 66 and 110 of gestation, only, relative to controls. This window of gestation coincides with the maximum placental growth and the start of exponential growth of the fetus when there are substantially increased metabolic demands on the placenta compared with earlier in gestation. Consequently, increased glucocorticoid sensitivity and suppressed IGF2 action could contribute to a switch in the placenta from proliferation to differentiation, thereby improving its nutrient transfer capacity. Upregulation of PPARG and UCP2 would promote placental fatty acid metabolism thereby limiting glucose utilization. These compensatory placental responses may serve to maintain fetal growth but could result in adverse adaptations such as the early onset of the metabolic syndrome in later life.

Accumulation of endocrine disrupting compounds in sheep fetal and maternal liver tissue following exposure to pastures treated with sewage sludge.

Fetal tissue concentrations of endocrine disrupting compounds (EDCs), and their relationship to maternal tissue concentrations, are largely unknown, in any species. In particular, the patterns of accumulation in the respective tissues following increased rates of environmental exposure are little known. This study was designed to determine fetal and maternal tissue concentrations of selected EDCs in sheep exposed to background, environmental concentrations of EDCs (pastures treated with inorganic fertiliser; Control; C) or to elevated, environmental concentrations (sludge-treated pastures; Treated; T). Mean log concentrations of diethylhexyl phthalate (DEHP) were similar in adult and fetal livers but there was a significant interaction between stage of development (maternal or fetal) and treatment reflecting the fact that mean concentrations were lower (P < 0.05) in C than T fetuses but not adults. Relative concentrations of polychlorinated biphenyls (PCB) in maternal and fetal tissue differed with congener; concentrations of congener 101 were higher (P < 0.05) in fetal tissue. Neither maternal nor fetal liver concentrations of any of the PCB congeners differed significantly with treatment. Polybrominated diphenyl ethers (PBDE) in the tissue were represented primarily by congeners 47 and 99. PBDE 99 concentrations were higher in maternal than fetal tissue (P = 0.01). None differed with treatment in either maternal or fetal tissues. Concentrations of many polycyclic aromatic hydrocarbons (PAH) were higher in maternal tissue but none differed with treatment in either adult or fetuses. It is concluded that sheep fetal liver EDC concentrations are variably related to those of their dams and in some cases appear to be selectively accumulated in fetuses. Differential accumulation of individual pollutants may have important implications for the assessment of risk from exposure.

Admissions processes at the seven United Kingdom veterinary schools: a review.

The major challenge in veterinary undergraduate admissions is to select those students with most suitability for veterinary training and careers from a large and diverse pool of applicants with very high academic ability. This paper describes a review of the admissions processes of the seven veterinary schools in the UK. There was significant commonality in the entry requirements and the criteria upon which the schools made decisions on candidates. There was some variation in the procedures used by individual schools to select candidates, but common themes existed within these processes. All of the schools evaluated both academic and non-academic factors for individual applicants, and all used interviews in some format as a selection tool after an initial short-listing process. The procedures and approaches to selection processes are compared and discussed.

Endocrine disruptors and other food-contaminating environmental pollutants as risk factors in animal reproduction.

Pollutants of many chemical classes, derived primarily from anthropogenic activities, are ubiquitous in the environment, persistent, biologically available and can exert adverse effects on the reproductive and other, indirectly related, physiological systems. Food is generally considered to be the major route of animal exposure in vertebrate species but the relative contributions of other routes of exposure such as through lungs, gills or skin are not well studied and may be of importance for certain animal groups, depending on their immediate environment. Animals are particularly sensitive to exposure during developmental stages but the pattern of exposure to chemicals is likely to be different to that of adults. Quantification of the risk posed by the ingestion of pollutants in food is complex and depends on many factors including species, diet composition, duration of exposure to the food, efficiency of pollutant absorption, subsequent metabolism, sensitivity of target organs and stage of development. While the effects of high doses of single chemicals are proven, dietary exposure to pollutants generally involves prolonged, low-level exposure to a large number of compounds, each of which has different chemical characteristics, exerts different biological effects and is present at varying concentrations. Thus, while exposure to pollutants through feed is undoubtedly a significant risk factor for many species and may be the most important one for many terrestrial vertebrates, other routes of exposure may be more important in other groups.

Maternal nutrient restriction in early pregnancy programs hepatic mRNA expression of growth-related genes and liver size in adult male sheep.

The liver is a major metabolic and endocrine organ of critical importance in the regulation of growth and metabolism. Its function is determined by a complex interaction of nutritionally regulated counter-regulatory hormones. The extent to which hepatic endocrine sensitivity can be programed in utero and whether the resultant adaptations persist into adulthood is unknown and was therefore the subject of this study. Young adult male sheep born to mothers that were fed either a control diet (i.e.100% of total live weight-maintenance requirements) throughout gestation or 50% of that intake (i.e. nutrient restricted (NR)) from 0 to 95 days gestation and thereafter 100% of requirements (taking into account increasing fetal mass) were entered into the study. All mothers gave birth normally at term, the singleton offspring were weaned at 16 weeks, and then reared at pasture until 3 years of age when their livers were sampled. NR offspring were of similar birth and body weights at 3 years of age when they had disproportionately smaller livers than controls. The abundance of mRNA for GH, prolactin, and IGF-II receptors, plus hepatocyte growth factor and suppressor of cytokine signaling-3 were all lower in livers of NR offspring. In contrast, the abundance of the mitochondrial protein voltage-dependent anion channel and the pro-apoptotic factor Bax were up regulated relative to controls. In conclusion, maternal nutrient restriction in early gestation results in adult offspring with smaller livers. This may be mediated by alterations in both hepatic mitogenic and apoptotic factors.

The effects of gene gun delivered pIL-3 adjuvant on skin pathology and cytokine expression.

The aim of this study was to investigate skin immunopathology following gene gun delivery of plasmid-encoding interleukin 3 (pIL-3) and hence explore the possible mechanisms of its adjuvant activity. Using the sheep as the experimental model, expressible pIL-3 was administered to the epidermis and the dermal/epidermal junction and its effects on the skin were assessed by histopathology, immunohistology and quantitative RT-PCR for a range of pro-inflammatory and immune response polarizing cytokines. Delivery of both functional and non-functional plasmids caused an acute inflammatory response with the infiltration of neutrophils and micro-abscess formation; however, the response to pIL-3 was more severe and was also associated with an early (24 h) infiltration of B cells and a later accumulation of CD172a-/CD45RA+ dendritic cells (DC). In terms of cytokine transcript expression, an early TNFalpha response was stimulated by gene gun delivery of plasmid-associated gold beads, which coincided with an immediate infiltration of neutrophils. However, only pIL-3 triggered the short-lived expression of IL-3 (peaking at 6 h) and significant long-term increases in both TNFalpha and IL-1beta. pIL-3 did not affect the expression of the immune response polarizing cytokines, IL-10 and IL-12.

Hypothalamic responses to peripheral glucose infusion in food-restricted sheep are influenced by photoperiod.

Nutritional feedback provided by systemic hormones, such as insulin and leptin, influences reproductive neuroendocrine output within the hypothalamus, yet the mechanisms and their interaction with photoperiodic cues remain unresolved in seasonal species. Here, peripheral glucose (G) infusion was used to increase endogenous concentrations of insulin and leptin in food-restricted sheep kept in either long-day (LD) or short-day (SD) photoperiod, and responses were examined in terms of pulsatile luteinising hormone (LH) (gonadotrophin-releasing hormone by inference) output and hypothalamic gene expression for nutritionally sensitive neuropeptides and receptors. We addressed the hypothesis that these hypothalamic responses were correlated and influenced by photoperiod. Oestradiol-implanted, castrated male sheep were kept 16 weeks in SD (8 h light/day) or LD (16 h light/day) and then transferred to the opposite photoperiods for 8 weeks, during which food was restricted to 90% requirement to maintain body weight (maintenance). For the final 6 days, food was reduced to 75% maintenance, and sheep in both photoperiods were infused intravenously with G (60 mM/h) or saline (S) (n = 8/group). G-infused sheep had higher mean plasma concentrations of G, insulin and leptin than S-infused sheep, with no effect of photoperiod. In LD, but not in SD, G infusion increased LH pulse frequency and pulse amplitude. In LD, but not in SD, gene expression in the hypothalamic arcuate nucleus was lower in G- than S-infused sheep for neuropeptide Y (NPY) and agouti-related peptide (AGRP) and was higher in G- than S-infused sheep for pro-opiomelanocortin (POMC). Gene expression for leptin and insulin receptors was not affected by photoperiod or infusion. These results are consistent with the involvement of NPY, AGRP and POMC in mediating the reproductive neuroendocrine response to increased systemic nutritional feedback, and they support the hypothesis that hypothalamic responses to nutritional feedback are influenced by photoperiod in sheep.

Diagnostic value of cytology of bronchoalveolar fluid for lung diseases of sheep.

Bronchoalveolar lavage fluid was collected postmortem from the lungs of 113 sheep, and total and differential cell counts were analysed in relation to the presence of gross and microscopic lung pathology. The diffuse lung diseases, maedi and adenomatosis, were both characterised by an increase in overall cellularity and by increases in the percentages of lymphocytes and neutrophils, respectively. Focal parasitic lung disease was characterised by an increase in the percentage of eosinophils and mast cells. Consolidated lung lesions were characterised by a slight increase in cellularity but no change in the differential cell profile. In regions of parasitised and consolidated lungs without lesions the differential cell profile was consistent with focal lung pathology, although the slight increase in cellularity observed in the consolidated regions was not observed in the regions without lesions. A decision tree was developed to facilitate the interpretation and indicate the likely predictive capacity of the differential cytology of the fluid.

Effect of the body condition of ewes on the secretion of LH and FSH and the pituitary response to gonadotrophin-releasing hormone.

The effects of body fat content (body condition) of ewes on hypothalamic activity and gonadotrophin-releasing hormone (GnRH) secretion and on pituitary sensitivity to GnRH were investigated using Scottish Blackface ewes. Two groups of 12 ewes were fed so that they achieved either a high body condition score (2.98, S.E.M. = 0.046; approximately 27% of empty body weight as fat) or a low body condition score (1.94, S.E.M. = 0.031; approximately 19% of empty body weight as fat) by 4 weeks before the period of study. Thereafter, they were differentially fed so that the difference in mean condition score was maintained. Oestrus was synchronized, and on day 11 of the subsequent cycle half of the ewes of each group were ovariectomized. On day 12, the remaining ewes were injected (i.m.) with 100 micrograms prostaglandin F2 alpha analogue and ovariectomized 30 h later. Numbers of large ovarian follicles and corpora lutea present at ovariectomy were recorded. Blood samples were collected at 15-min intervals for 12 h on day 10 of the cycle (luteal phase) and at 10-min intervals from 24 to 30 h after prostaglandin injection (follicular phase). At days 2 and 7 after ovariectomy, samples were collected at 15-min intervals for 8 h and ewes were then injected with 10 micrograms GnRH and samples were collected for a further 3 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of level of food intake of ewes on the secretion of LH and FSH and on the pituitary response to gonadotrophin-releasing hormone in ovariectomized ewes.

The effect of level of food intake on LH and FSH profiles and pituitary sensitivity to gonadotrophin-releasing hormone (GnRH) was investigated in two groups of 12 ovariectomized ewes. Ewes with a high intake (group H) had a mean daily intake (+/- S.E.M.) of 1.99 +/- 0.075 kg dry matter (DM)/head per day while ewes with a moderate intake (group M) consumed a mean of 1.02 +/- 0.021 kg DM/head per day. Ovaries were surgically removed from six ewes of each group on day 11 of the luteal phase and from the remainder 30 h after an injection of 100 micrograms prostaglandin analogue given on day 11 to induce luteolysis. During both the luteal phase and the follicular phase, mean LH and FSH concentrations and LH pulse frequencies and amplitudes were unaffected by the level of intake but mean plasma prolactin concentrations were higher (P less than 0.05) in group H than in group M ewes in the follicular phase. Mean LH and FSH concentrations at day 2 after ovariectomy were unaffected by treatment while mean prolactin concentrations were higher (P less than 0.05) in group H than in group M ewes. At day 7 after ovariectomy, mean LH and FSH concentrations were lower (P less than 0.05) in group H than in group M ewes although mean LH pulse frequencies and pulse amplitudes were not significantly affected by the level of intake at either time.(ABSTRACT TRUNCATED AT 250 WORDS)

Maternal undernutrition alters triiodothyronine concentrations and pituitary response to GnRH in fetal sheep.

The aims of this study were to determine which hormones may have a role in the expression of maternal undernutrition effects on reproductive function, in both the developing fetus and the adult offspring. This was undertaken by measuring the effects of long-term maternal undernutrition on metabolic hormone profiles and pituitary responses to single doses of GnRH and GH-releasing factor (GRF) in fetal sheep. From mating, groups of ewes were fed rations providing either 100% (HIGH) or 50% (LOW) of estimated metabolisable energy requirements for pregnancy throughout the experiment until slaughter at approximately 119 days of gestation. Fetal and maternal blood samples were collected from 113 until 119 days of gestation, via carotid and jugular catheters respectively, and assayed for insulin, IGF-I, GH, thyroxine and triiodothyronine (T(3)). Undernutrition had no effects on fetal weight, fetal gonad weight of either sex, fetal insulin or IGF-I concentrations. Male LOW fetuses exhibited a significantly attenuated response (P<0.05) to a bolus challenge of GnRH compared with HIGH fetuses. Basal fetal GH concentrations and the response to exogenous GRF were similar in both treatment groups, although LOW fetuses exhibited more secretory episodes (P<0.01). Mean T(3) concentrations were significantly lower in both the maternal (P<0.01) and fetal (P<0.05) plasma of LOW animals compared with HIGH animals. It is concluded that pituitary function was altered in fetal males and could influence male reproductive development. On the other hand, in female sheep, fetal gonadal abnormalities and reductions in reproductive capacity in adult life which are associated with fetal undernutrition are unlikely to be attributable to altered pituitary function. Additionally, these studies raise the possibility that thyroid hormones may have a role in the expression of maternal undernutrition effects on fetal development.

Effects of nutritional status and gonadal steroids on expression of appetite-regulatory genes in the hypothalamic arcuate nucleus of sheep.

Sheep exhibit photoperiod-driven seasonal changes in appetite and body weight so that nutritional status increases in long days (LD) and decreases in short days (SD); additionally, they are reproductively active in SD and inactive in LD. We addressed the hypothesis that appetite-regulatory genes in the hypothalamus respond differently to changes in nutritional feedback induced by photoperiod as opposed to food restriction, and that responses would be influenced by gonadal steroid status. Castrated oestradiol-implanted male sheep were kept in SD (8 h light/day) or LD (16 h light/day) for 11 weeks, with ad libitum or restricted food (experiment 1; n=8/group). Rams were kept in SD or LD for 12 weeks with ad libitum or restricted food (experiment 2; n=6/group). Gene expression (by in situ hybridisation) in the hypothalamic arcuate nucleus for leptin receptor (OB-Rb), neuropeptide Y (NPY), pro-opiomelanocortin (POMC) and agouti-related peptide (AGRP) was unaffected by photoperiod treatment, but food restriction increased NPY and AGRP mRNAs, in experiment 1. In experiment 2, mRNAs for POMC and cocaine- and amphetamine-regulated transcript (CART) were up-regulated and AGRP down-regulated in SD, while food restriction increased OB-Rb mRNA, increased NPY and AGRP mRNAs only in LD and decreased POMC mRNA only in SD. Thus, gene expression responded differently to photoperiod and food restriction, and the melanocortin pathway was up-regulated in SD in reproductively activated rams but not in oestradiol-implanted castrates. These data support the hypothesis that hypothalamic appetite-regulatory pathways respond differently to changes in nutritional feedback induced by photoperiod as opposed to food restriction, with gonadal steroid feedback additionally influencing the responses.

Contrasting effects of different levels of food intake and adiposity on LH secretion and hypothalamic gene expression in sheep.

Body reserves (long-term) and food intake (short-term) both contribute nutritional feedback to the hypothalamus. Reproductive neuroendocrine output (GnRH/LH) is stimulated by increased food intake and not by high adiposity in sheep, but it is unknown whether appetite-regulating hypothalamic neurons show this differential response. Castrated male sheep (Scottish Blackface) with oestradiol implants were studied in two 4 week experiments. In Experiment 1, sheep were fed to maintain the initial body condition (BC) score of 2.0+/-0.00 (lower BC (LBC), n=7) or 2.9+/-0.09 (higher BC (HBC), n=9), and liveweight of 43+/-1.1 and 59+/-1.6 kg respectively. LBC and HBC sheep had similar mean plasma LH concentration, pulse frequency and amplitude, but HBC animals had higher mean plasma concentrations of insulin (P<0.01), leptin (P<0.01) and glucose (P<0.01). Gene expression (measured by in situ hybridisation) in the hypothalamic arcuate nucleus (ARC) was higher in LBC than HBC sheep for neuropeptide Y (NPY; 486% of HBC, P<0.01), agouti-related peptide (AGRP; 467%, P<0.05) and leptin receptor (OB-Rb; 141%, P<0.05), but lower for cocaine- and amphetamine-regulated transcript (CART; 92%, P<0.05) and similar between groups for pro-opiomelanocortin (POMC). In Experiment 2, sheep with initial mean BC score 2.4+/-0.03 and liveweight 55+/-0.8 kg were fed a liveweight-maintenance ration (low intake, LI, n=7) while sheep with initial mean BC score 2.0+/-0.03 and liveweight 43+/-1.4 kg were fed freely so that BC score increased to 2.5+/-0.00 and liveweight increased to 54+/-1.4 kg (high intake, HI, n=9). Compared with LI, HI sheep had higher mean plasma LH (P<0.05), baseline LH (P<0.01) and pulse amplitude (P<0.01) and showed a trend towards higher pulse frequency. Although there were no differences in final mean plasma concentrations, there were significant increases over time in mean concentrations of insulin (P<0.001), leptin (P<0.05) and glucose (P<0.001) in HI sheep. Gene expression for AGRP in the ARC was higher in HI than LI animals (453% of LI; P<0.05), but expression levels were similar for NPY, OB-Rb, CART and POMC. Thus, the hypothalamus shows differential responses to steady-state adiposity as opposed to an increase in food intake, in terms of both reproductive neuroendocrine activity and hypothalamic appetite-regulating pathways. Differences in hypothalamic gene expression were largely consistent with contemporary levels of systemic leptin and insulin feedback; however, increased nutritional feedback was stimulatory to GnRH/LH whereas constant high feedback was not. The hypothalamus therefore has the ability to retain a nutritional memory that can influence subsequent responses.

Effect of maternal overnutrition during pregnancy on pituitary gonadotrophin gene expression and gonadal morphology in female and male foetal sheep at day 103 of gestation.

The aim was to determine whether nutritionally mediated restriction of placental growth alters foetal body growth, pituitary gonadotrophin gene expression and gonadal development at Day 103 of gestation. Embryos recovered from adult ewes inseminated by a single sire were transferred, singly, into the uteri of adolescent recipients. After transfer, adolescent ewes were offered a high (H, n=16) or moderate (M, n=12) level of a complete diet. Ewes were slaughtered at 103+/-0.2 days of gestation and foetal blood, brain, pituitary and gonads were collected. Mean placental weight was lower (P< 0.01) in H than in M groups but foetal weight and reproductive organ weights were similar. Maternal nutrition did not influence LHbeta or FSHbeta mRNA expression in either sex but FSHbeta mRNA expression was higher (P< 0.001) in female (n=11) than in male (n=17) foetal pituitaries. Mean foetal plasma gonadotrophin concentrations were not influenced by dietary intake in either sex. Plasma progesterone concentrations were lower (P=0.001) in foetuses derived from H compared with M intake dams. Compared with M foetuses (n=5), ovaries from H foetuses (n=6) had fewer primordial follicles (P< 0.05) and fewer follicles in total (P< 0.005). In contrast, maternal nutritional status did not influence either seminiferous cord or Sertoli cell numbers in male foetuses (H, n=10; M, n=7). It is concluded that high maternal nutrient intakes restricted placental growth and altered foetal ovarian follicular development prior to the end of the second third of gestation. The latter effect was independent of gonadotrophin secretion.Crown

Effects of Beta-endorphin on pulsatile luteinizing hormone and prolactin secretion during the follicular phase in the ewe.

Abstract The present study was undertaken to determine the effects of the endogenous opioid ligandbeta-endorphin on pulsatile luteinizing hormone (LH) secretion and plasma prolactin concentrations during the follicular phase of the ewe. Oestrous cycles were synchronized by injection of prostaglandin analogue and, commencing 13 h later, saline or beta-endorphin (2, 10 or 50 mug) was injected intracerebroventricularly at hourly intervals for 3 h. Treatment with beta-endorphin was followed by a significant reduction in LH pulse frequency at all doses due to almost complete cessation of pulses. There were no significant changes in LH pulse amplitude or mean LH concentrations. At the lowest dose ofbeta-endorphin, LH pulses recommenced within 3 h of the last injection in all animals and pulse frequency was not significantly different from the saline-injected controls during the 3 h post-treatment period. Following treatment with 10 or 50 mug beta-endorphin, LH pulse frequency remained suppressed during the 3 h post-treatment period but was not different from saline-treated controls on the following day. The time to the onset of the LH surge was not affected by intracerebroventricularbeta-endorphin. Plasma prolactin concentrations were significantly increased following intracereb-roventricular injection of 10 or 50 mug beta-endorphin, declining to control values soon after treatments stopped. Intravenous administration of 50 mug beta-endorphin had no effect on LH but was accompanied by a small increase in prolactin concentrations. While these results indicate that hypothalamicbeta-endorphin may be involved in the central control of LH and prolactin secretion, they provide no evidence for subtle modulation of LH pulse frequency by this neuropeptide.

Appetite regulatory neuropeptides are expressed in the sheep hypothalamus before birth.

In the adult, a hypothalamic neural network acts to maintain energy balance in response to nutritional feedback from the periphery. Although there is an immediate requirement for this system to be functional at birth, it is unknown whether the components of this central neural network are expressed in the developing brain before birth. We therefore examined in the fetal sheep hypothalamus during late gestation gene expression for leptin receptor (OB-Rb) and neuropeptides that regulate energy balance in the adult. Brains were collected from fetal sheep at 110 days (n = 12) and 140 days of gestation (n = 5) (term = 150 days) and gene expression was detected in all hypothalami using in situ hybridization with radiolabelled riboprobes for OB-Rb, neuropeptide Y (NPY), agouti-related peptide, pro-opiomelanocortin and cocaine- and amphetamine-regulated transcript (CART). All mRNAs were expressed in the arcuate nucleus of fetuses at both time points. Additional sites of mRNA expression were the dorsomedial hypothalamus (DMH) for NPY, the paraventricular nucleus (PVN), ventromedial hypothalamus (VMH) and lateral hypothalamic area for CART, and the DMH, PVN and VMH for OB-Rb. We have therefore demonstrated that adult-like localization of gene expression for OB-Rb and key appetite regulatory neuropeptides is established in the ovine hypothalamus before birth. Thus, the fetus possesses a central appetite regulatory neural network with the potential to respond to changes in nutrient supply, which could impact on energy balance regulation both before and after birth.