Reproduction Symposium: does grazing on biosolids-treated pasture pose a pathophysiological risk associated with increased exposure to endocrine disrupting compounds?

Biosolids (processed human sewage sludge), which contain low individual concentrations of an array of contaminants including heavy metals and organic pollutants such as polycyclic aromatic hydrocarbons (PAH), polychlorinated biphenyls (PCB), and polychlorinated dibenzodioxins/polychlorinated dibenzofurans known to cause physiological disturbances, are increasingly being used as an agricultural fertilizer. This could pose a health threat to both humans and domestic and wild animal species. This review summarizes results of a unique model, used to determine the effects of exposure to mixtures of environmentally relevant concentrations of pollutants, in sheep grazed on biosolids-treated pastures. Pasture treatment results in nonsignificant increases in environmental chemical (EC) concentrations in soil. Whereas EC concentrations were increased in some tissues of both ewes and their fetuses, concentrations were low and variable and deemed to pose little risk to consumer health. Investigation of the effects of gestational EC exposure on fetal development has highlighted a number of issues. The results indicate that gestational EC exposure can adversely affect gonadal development (males and females) and that these effects can impact testicular morphology, ovarian follicle numbers and health, and the transcriptome and proteome in adult animals. In addition, EC exposure can be associated with altered expression of GnRH, GnRH receptors, galanin receptors, and kisspeptin mRNA within the hypothalamus and pituitary gland, gonadotroph populations within the pituitary gland, and regional aberrations in thyroid morphology. In most cases, these anatomical and functional differences do not result in altered peripheral hormone concentrations or reproductive function (e.g., lambing rate), indicating physiological compensation under the conditions tested. Physiological compensation is also suggested from studies that indicate that EC effects may be greater when exposure occurs either before or during gestation compared with EC exposure throughout life. With regard to human and animal health, this body of work questions the concept of safe individual concentration of EC when EC exposure typically occurs as complex mixtures. It suggests that developmental EC exposure may affect many different physiological systems, with some sex-specific differences in EC sensitivity, and that EC effects may be masked under favorable physiological conditions.

Foetal hypothalamic and pituitary expression of gonadotrophin-releasing hormone and galanin systems is disturbed by exposure to sewage sludge chemicals via maternal ingestion.

Animals and humans are chronically exposed to endocrine disrupting chemicals (EDCs) that are ubiquitous in the environment. There are strong circumstantial links between environmental EDC exposure and both declining human/wildlife reproductive health and the increasing incidence of reproductive system abnormalities. The verification of such links, however, is difficult and requires animal models exposed to 'real life', environmentally relevant concentrations/mixtures of environmental contaminants (ECs), particularly in utero, when sensitivity to EC exposure is high. The present study aimed to determine whether the foetal sheep reproductive neuroendocrine axis, particularly gondotrophin-releasing hormone (GnRH) and galaninergic systems, were affected by maternal exposure to a complex mixture of chemicals, applied to pasture, in the form of sewage sludge. Sewage sludge contains high concentrations of a spectrum of EDCs and other pollutants, relative to environmental concentrations, but is frequently recycled to land as a fertiliser. We found that foetuses exposed to the EDC mixture in utero through their mothers had lower GnRH mRNA expression in the hypothalamus and lower GnRH receptor (GnRHR) and galanin receptor (GALR) mRNA expression in the hypothalamus and pituitary gland. Strikingly, this, treatment had no significant effect on maternal GnRH or GnRHR mRNA expression, although GALR mRNA expression within the maternal hypothalamus and pituitary gland was reduced. The present study clearly demonstrates that the developing foetal neuroendocrine axis is sensitive to real-world mixtures of environmental chemicals. Given the important role of GnRH and GnRHR in the regulation of reproductive function, its known role programming role in utero, and the role of galanin in the regulation of many physiological/neuroendocrine systems, in utero changes in the activity of these systems are likely to have long-term consequences in adulthood and represent a novel pathway through which EC mixtures could perturb normal reproductive function.

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.

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.

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.

Environmental influences on the fetus and neonate--timing, mechanisms of action and effects on subsequent adult function.

Environmental influences on fetal and neonatal development can affect neural, reproductive, immune and cardiovascular function in adult humans and animals. The effects can be exerted at many different stages of development from before conception to after birth. Effects may even be exerted during a preceding generation. Some known and some possible mechanisms are reviewed. Systems likely to be affected include the brain, hypothalamus, pituitary and adrenal glands and the gonads. The effects may be exerted through altered gene expression at any stage of development or through changes in organ structure or physiology.

Undernutrition of ewe lambs in utero and in early post-natal life does not affect hypothalamic-pituitary function in adulthood.

The effect of undernutrition in utero, during late gestation (from day 100), and early neonatal life on hypothalamic-pituitary function was investigated in female lambs born to ewes fed rations calculated to provide either 100% (high; H) or 70% (low; L) of the energy requirements to sustain a twin pregnancy. Following parturition in early spring, ewes and lambs were maintained on pasture with sward heights of 6 cm (H) or 4 cm (L) until week 8 of lactation and then sward heights of 5 cm (H) or 3 cm (L) until weaning at week 14. Mean lamb birth weights were 18% lower in L than H animals (P<0.05) and mean liveweights were 23% lower in the L animals (P<0.001) at weaning at 14 weeks of age. Liveweight differences were not significant at, or after, 26 weeks of age. There were no significant differences between pre-pubertal H and L animals, either before (26 weeks) or after ovariectomy (31 weeks), with respect to hypothalamic or pituitary activity, as measured by LH pulse frequency, pulse amplitude or mean plasma LH and FSH concentrations and the responses to GnRH injection as measured by LH peak amplitude, respectively. Similarly there were no differences in any of these variables in pubertal animals at 18 months of age. At 31 weeks of age, H animals had significantly lower pituitary GnRH receptor binding (P<0.01) and lower ERalpha mRNA content (P<0.05) than L lambs. There were no differences with treatment in the abundance of mRNA for LHbeta, FSHbeta or GnRH-receptor at 31 weeks of age or in pubertal animals aged 18 months, when there were no significant differences with treatment in GnRH receptor binding or ERalpha mRNA expression. It is concluded that effects on lifetime reproductive function of female sheep of undernutrition during late gestation and early neonatal life are unlikely to be expressed through permanent changes in hypothalamic-pituitary function and are therefore attributable to effects exerted directly on the ovary.

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.