Epigenetics and developmental programming of welfare and production traits in farm animals.

The concept that postnatal health and development can be influenced by events that occur in utero originated from epidemiological studies in humans supported by numerous mechanistic (including epigenetic) studies in a variety of model species. Referred to as the 'developmental origins of health and disease' or 'DOHaD' hypothesis, the primary focus of large-animal studies until quite recently had been biomedical. Attention has since turned towards traits of commercial importance in farm animals. Herein we review the evidence that prenatal risk factors, including suboptimal parental nutrition, gestational stress, exposure to environmental chemicals and advanced breeding technologies, can determine traits such as postnatal growth, feed efficiency, milk yield, carcass composition, animal welfare and reproductive potential. We consider the role of epigenetic and cytoplasmic mechanisms of inheritance, and discuss implications for livestock production and future research endeavours. We conclude that although the concept is proven for several traits, issues relating to effect size, and hence commercial importance, remain. Studies have also invariably been conducted under controlled experimental conditions, frequently assessing single risk factors, thereby limiting their translational value for livestock production. We propose concerted international research efforts that consider multiple, concurrent stressors to better represent effects of contemporary animal production systems.

Adiposity and plane of nutrition influence reproductive neuroendocrine and appetite responses to intracerebroventricular insulin and neuropeptide-Y in sheep.

Long-term nutritional background is thought to influence hypothalamic appetite and reproductive neuroendocrine responses to short-term nutritional feedback. In order to investigate this phenomenon, the effects of intracerebroventricular administration of insulin or neuropeptide-Y (NPY) on LH secretion and voluntary food intake (VFI) were examined in sheep that were initially thin and kept on an increasing nutritional plane (INP), or initially fat and kept on a decreasing nutritional plane (DNP), for 10 weeks. Intracerebroventricular insulin stimulated LH secretion and suppressed VFI in INP sheep when initially thin, but not when they became fat, and had no effect on LH in DNP sheep when initially fat, and stimulated LH secretion when they became thin. Intracerebroventricular NPY had no effect on LH or VFI in INP sheep when initially thin, decreased LH secretion and increased VFI when they became fat, and decreased LH secretion in DNP sheep when initially fat but had no effect when they became thin. Therefore, sensitivity to insulin increases with low or decreasing nutritional status and decreases with high or increasing nutritional status, whereas sensitivity to NPY increases with high or increasing nutritional status and decreases with low or decreasing nutritional status. In conclusion, reproductive neuroendocrine and appetite responses to acute changes in nutritional feedback signals depend on the individual's longer-term nutritional background.

Decreased blood-brain leptin transfer in an ovine model of obesity and weight loss: resolving the cause of leptin resistance.

OBJECTIVE: Hypothalamic resistance to the anorexigenic actions of the peripheral adipostat hormone leptin is characteristic of obesity. Here, we use an obese animal model of similar body weight to that of the human to test in vivo whether leptin resistance is due to decreased blood-brain leptin transport or intra-hypothalamic insensitivity, and whether sensitivity to leptin is restored by weight loss. For 40 weeks, adult sheep surgically prepared with intra-cerebroventricular (ICV) cannulae were given a complete natural diet ad libitum ('Obese' group) or in restricted quantities ('Lean' group), and then the dietary amounts were reversed for 16 weeks until mean group body weights converged ('Slimmers' and 'Fatteners', respectively). RESULTS: ICV leptin injection (0.5 mg) at 8-week intervals acutely decreased voluntary food intake by approximately 35% in the 'Obese' group on each occasion and in 'Slimmers' and 'Fatteners' at the end, providing no evidence of intra-hypothalamic insensitivity. The ratio between endogenous leptin concentrations in ventricular cerebrospinal fluid (CSF) and peripheral blood decreased with increasing leptinaemia in 'Obese' sheep, indicating decreased efficiency of blood-brain leptin transport, whereas leptin concentrations remained low and the CSF:blood ratio remained high in 'Lean' sheep. Compared with 'Fatteners' of similar body weight, 'Slimmers' were hypoleptinaemic, but their CSF:blood leptin concentration ratio remained low. Thus, the obesity-induced impairment of leptin blood-brain transport was sustained despite an approximately 15% weight loss. CONCLUSION: These results support the hypothesis that central resistance to leptin in obesity with associated peripheral hyperleptinaemia is attributable to decreased efficiency of leptin transport into the brain and not to intra-hypothalamic leptin insensitivity. However, leptin transport efficiency is not restored after weight loss by caloric restriction despite the prevailing hypoleptinaemia.

Increased maternal nutrition alters development of the appetite-regulating network in the brain.

Individuals exposed to an increased nutrient supply before birth have a high risk of becoming obese children and adults. It has been proposed that exposure of the fetus to high maternal nutrient intake results in permanent changes within the central appetite regulatory network. No studies, however, have investigated the impact of increased maternal nutrition on the appetite regulatory network in species in which this network develops before birth, as in the human. In the present study, pregnant ewes were fed a diet which provided 100% (control, n = 8) or approximately 160% (well-fed, n = 8) of metabolizable energy requirements. Ewes were allowed to lamb spontaneously, and lambs were sacrificed at 30 days of postnatal age. All fat depots were dissected and weighed, and expression of the appetite-regulating neuropeptides and the leptin receptor (OBRb) were determined by in situ hybridization. Lambs of well-fed ewes had higher glucose (Glc) concentrations during early postnatal life (F = 5.93, P<0.01) and a higher relative subcutaneous (s.c.) fat mass at 30 days of age (34.9+/-4.7 g/kg vs. 22.8+/-3.3 g/kg; P<0.05). The hypothalamic expression of pro-opiomelanocortin was higher in lambs of well-fed ewes (0.48+/-0.09 vs. 0.28+/-0.04, P<0.05). In lambs of overnourished mothers, but not in controls, the expression of OBRb was inversely related to total relative fat mass (r2 = 0.50, P = 0.05, n = 8), and the direct relationship between the expression of the central appetite inhibitor CART and fat mass was lost. The expression of neuropeptide Y and AGRP was inversely related to total relative fat mass (NPY, r2 = 0.28, P<0.05; agouti-related peptide, r2 = 0.39, P<0.01). These findings suggest that exposure to increased nutrition before birth alters the responses of the central appetite regulatory system to signals of increased adiposity after birth.

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.

Hepatic IGF1 DNA methylation is influenced by gender but not by intrauterine growth restriction in the young lamb.

Intrauterine growth restriction (IUGR) and postnatal catch-up growth confer an increased risk of adult-onset disease. Overnourishment of adolescent ewes generates IUGR in ∼ 50% of lambs, which subsequently exhibit increased fractional growth rates. We investigated putative epigenetic changes underlying this early postnatal phenotype by quantifying gene-specific methylation at cytosine:guanine (CpG) dinucleotides. Hepatic DNA/RNA was extracted from IUGR [eight male (M)/nine female (F)] and normal birth weight (12 M/9 F) lambs. Polymerase chain reaction was performed using primers targeting CpG islands in 10 genes: insulin, growth hormone, insulin-like growth factor (IGF)1, IGF2, H19, insulin receptor, growth hormone receptor, IGF receptors 1 and 2, and the glucocorticoid receptor. Using pyrosequencing, methylation status was determined by quantifying cytosine:thymine ratios at 57 CpG sites. Messenger RNA (mRNA) expression of IGF system genes and plasma IGF1/insulin were determined. DNA methylation was independent of IUGR status but sexual dimorphism in IGF1 methylation was evident (MF (both P<0.001). IGF1 mRNA expression correlated negatively with IGF1 methylation (r=-0.507, P=0.002) and positively with plasma IGF1 (r=0.884, P<0.001). Carcass and empty body weights were greater in males (P=0.002-0.014) and this gender difference in early body conformation was mirrored by sexual dimorphism in hepatic IGF1 DNA methylation, mRNA expression and plasma IGF1 concentrations.

Orexigenic Gene Expression in Late Gestation Ovine Foetal Hypothalamus is Sensitive to Maternal Undernutrition and Realimentation.

Adverse nutritional effects on developing foetal hypothalamic appetitive pathways may contribute to programmed hyperphagia and obesity in intra-uterine growth-restricted, low birth weight offspring. In the present study, for the first time, hypothalamic gene expression for primary orexigenic and anorexigenic genes was examined in late gestation ovine foetuses (130 days; term=145 days) whose mothers were undernourished (UN) or well-nourished (C) throughout pregnancy, or transferred from UN to C on day 90 (UN-C). Pregnancies resulted from singleton embryo transfer into adolescent growing ewes. Body weight, carcass fat content and perirenal adipose tissue (PAT) mass were all lower for UN (n=9) than C (n=7) and intermediate for UN-C foetuses (n=6), with no effect of sex. PAT leptin gene expression (by the reverse transcriptase-polymerase chain reaction) was lower in UN than C and UN-C groups, and lower in males than females. Gene expression (by in situ hybridisation with radiolabelled riboprobes) in the arcuate nucleus was greater in UN than C foetuses for neuropeptide Y (NPY), agouti-related peptide (AGRP) and leptin receptor (OBRb) but not different for pro-opiomelanocortin and cocaine- and amphetamine-regulated transcript. Gene expression in UN-C foetuses was intermediate for NPY and AGRP and not different from C foetuses for OBRb. Gene expression for NPY, AGRP and OBRb correlated negatively with foetal carcass fat content and with PAT leptin gene expression across all groups. Males had greater mRNA expression for AGRP than females, with NPY and OBRb showing similar trends. Therefore, maternal undernutrition throughout pregnancy increased orexigenic gene expression in the late gestation foetal hypothalamus, and expression levels were largely normalised by improved maternal nutrition in the last third of pregnancy. These findings may have implications for avoiding or correcting prenatal programming of postnatal hyperphagia and obesity.

Photoperiod regulates growth, puberty and hypothalamic neuropeptide and receptor gene expression in female Siberian hamsters.

In seasonal mammals, both the growth and reproductive axes are regulated by photoperiod. Female Siberian hamsters were kept, for up to 12 weeks, in long-day (LD) or short-day (SD) photoperiod, from weaning at 3 weeks of age (Exp 1). LD hamsters had characteristically faster growth and higher asymptotic body weight, adiposity, and leptin gene expression in adipose tissue. Only LD females attained puberty. Gene expression in the hypothalamic arcuate nucleus for leptin receptor (OB-Rb), POMC, and melanocortin 3-receptor (MC3-R) was higher in LD but did not change from weaning levels in SD. In contrast, gene expression in the arcuate nucleus for cocaine and amphetamine-regulated transcript (CART) was higher in SD than LD, a difference that was apparent at 2 weeks post weaning. Transfer of SD females to LD at 15 weeks post weaning (Exp 2) increased body weight, leptin signal, and gene expression for POMC but failed to induce normal puberty onset or to increase gene expression for OB-Rb and MC3-R. Therefore, photoperiodic regulation of puberty may be modulated by age, by photoperiodic history, and by changes in leptin signaling and the activity of the leptin-sensitive hypothalamic melanocortin system (POMC, MC3-R). A role for CART in photoperiodic regulation of growth is suggested, because the changes in CART gene expression preceded significant divergence of growth trajectories in the opposite photoperiods.

Seasonally inappropriate body weight induced by food restriction: effect on hypothalamic gene expression in male Siberian hamsters.

Male Siberian hamsters undergo physiological weight change in changing photoperiod. Weight loss was induced by food restriction in long days to mimic short-day weight loss, or by food restriction superimposed on short-day weight loss, to test the hypothesis that the hypothalamus differentiates between weight change induced by imposed negative energy balance (inappropriate body weight) and seasonal, appropriate, body weight change, even when these are of similar magnitude. Short-day weight loss was accompanied by reduced POMC and leptin receptor (OB-Rb) mRNA in the arcuate nucleus but elevated cocaine- and amphetamine-regulated transcript. Melanocortin 3-receptor gene expression was reduced in the arcuate nucleus but elevated in the ventromedial nucleus compared with ad libitum-fed long-day controls. Weight loss in long-day restricted animals generated a gene expression profile typical of negative energy balance with low cocaine- and amphetamine-regulated transcript mRNA and elevated OB-Rb. Melanocortin 3-receptor mRNA levels were indistinguishable in short-day and long-day food-restricted hamsters. The hypothalamic correlates of food restriction in short days included up-regulated anabolic neuropeptides and increased OB-Rb mRNA. Low plasma leptin is integrated differently in short-day and long-day restricted animals, and seasonally-inappropriate body weight in either photoperiod engages the compensatory neuropeptide systems involved in the defense of body weight.

Daily patterns of plasma leptin in sheep: effects of photoperiod and food intake.

Circulating concentrations of leptin in sheep correlate with body fatness and are affected by level of food intake and photoperiod. The present objective was to elucidate the short-term dynamics of leptin secretion. Frequent blood samples were taken over 48 h from 12 Soay rams after 16 weeks in short-day photoperiod (SD, 16 h darkness:8 h light) with freely available food, and then after 16 weeks in long days (16 h light:8 h darkness) with food freely available (LD) or restricted to 90% maintenance (LDR) (n=6/group). During the second 24 h of sampling, half were food deprived (n=6, SD and LD) and half had their meal times shifted (n=6, SD and LDR). A homologous RIA was developed, using antibodies raised in chicken against recombinant ovine leptin, to measure plasma concentrations. Simultaneous 24 h profiles of plasma insulin, glucose and non-esterified fatty acids (NEFA) were measured. Plasma leptin was higher in LD than SD, and in LD than LDR, associated with higher food intake, liveweight and body condition score (adiposity), but tended to be lower in LDR than SD, associated with lower food intake, liveweight and body condition score. There was no evidence for a circadian rhythm of plasma leptin, but clear evidence for post-prandial peaks of low amplitude (15-36%) 2-8 h after meals given at normal and shifted times. Complete food deprivation caused a dramatic fall in plasma leptin to basal levels within 24 h. There was a positive association of plasma leptin with plasma insulin, and negative association with NEFA, both between meals and during fasting. Thus, plasma leptin concentrations in sheep are sensitive to short-term changes in energy balance, as well as to long-term photoperiod-driven changes in food intake and adiposity.

Seasonal and dose-dependent effects of intracerebroventricular leptin on lh secretion and appetite in sheep.

The role of leptin in neuroendocrine appetite and reproductive regulation remains to be fully resolved. A series of three experiments was conducted using adequately nourished oestradiol-implanted castrated male sheep. In a cross-over design (n=6), responses to a single i.c.v. (third ventricle) injection of leptin (0.5, 1.0 and 1.5 mg ovine leptin (oLEP) and 1.0 mg murine leptin (mLEP)), N-methyl-D-aspartate (NMDA, 20 micro g) or 0.9% saline (control) were measured in terms of LH secretion (4 h post-injection compared with 4 h pre-injection) and appetite (during 2 h post-injection) in autumn (Experiment 1). NMDA and 1.0 mg oLEP treatments were repeated in the same sheep in the following spring (Experiment 2). With an additional 12 sheep (n=18 in cross-over design), responses to low-dose 'physiological' i.c.v. infusion of leptin (8 ng/h for 12 h daily for 4 days), insulin (0.7 ng/h) and artificial cerebrospinal fluid were measured in the next spring (Experiment 3). LH was studied over 8 h and appetite over 1 h on days 1 and 4 of infusion. In Experiment 1 (autumn), oLEP overall increased LH pulse frequency by up to 110% (P<0.05), decreased LH pulse amplitude (P<0.05) and decreased appetite (P<0.05). mLEP reduced LH pulse amplitude (P<0.05) without significant effect on appetite, while NMDA reduced appetite (P<0.05) but had no effect on LH. In Experiment 2 (spring), LH responses were 'surge-like' with highly significant increases in the moving average LH concentration after 1.0 mg oLEP (P<0.001) and after NMDA (P<0.001). Compared with similar analysis of experiment 1 results, the LH response in spring was greater than that in autumn for both 1.0 mg oLEP (P<0.05) and NMDA (P<0.005). Conversely, unlike in autumn (Experiment 1), there was no effect of 1.0 mg oLEP or NMDA on appetite in the spring (Experiment 2). In Experiment 3 (spring), 'physiological' i.c.v. infusion of oLEP or insulin increased LH pulse frequency by up to 100% (P<0.001) compared with the control infusion on both days 1 and 4, but there were no effects on appetite. These results indicate that intracerebral leptin both stimulates reproductive neuroendocrine output and decreases appetite in adequately nourished sheep. However, the responses of these two axes were dose-dependent and differentially affected by the time of year, suggesting dissociation of the neural pathways involved.

Effect of chronic food restriction on pulsatile luteinizing hormone secretion and hypothalamic neuropeptide Y gene expression in castrate male sheep.

Castrate male sheep (wethers, average liveweight 38 +/- 0.6 kg) were given one of the following diets for 10 weeks followed by euthanasia (n = 8/group): high-energy high-protein providing 1-5 times the energy required to maintain liveweight (maintenance) (group 1.5 M), low-energy low-protein at 0.5 maintenance (0.5 M), or low-energy high-protein at 0.5 maintenance (0.5 M + P). 1.5 M wethers gained 22% liveweight whereas 0.5 M and 0.5 M + P wethers lost 18 and 13% liveweight respectively. Relative to the 1.5 M group, the 0.5 M and 0.5 M + P groups had similar plasma concentrations of glucose and cortisol throughout, but elevated non-esterified fatty acids (P < 0.001) and reduced IGF-I and insulin (P < 0.05, 0.01 or 0.001) from 1 week onwards. Each week blood samples were taken every 12 min for 4 h and plasma assayed for LH. Mean concentration over 4 h, LH pulse frequency and LH pulse amplitude showed no progressive change in 1.5 M sheep. However, in both 0.5 M and 0.5 M + P groups mean LH increased (P < 0.001 and P < 0.01 respectively), pulse frequency decreased (P < 0.01 and P < 0.01) and pulse amplitude increased (P < 0.001 and P < 0.01) over the 10-week period. Anterior pituitary. LH content was greater in 0.5 M (P < 0.01) and 0.5 M + P (P < 0.05) than in 1.5 M sheep. Coronal sections (20 microns) of hypothalamic brain tissue were subjected to in situ hybridisation to determine gene expression for neuropeptide Y (NPY). NPY mRNA was concentrated in the arcuate nucleus and median eminence, with total amounts greater in both 0.5 M (310%, P < 0.001) and 0.5 M + P (333%, P < 0.01) groups than in 1.5 M sheep (100%). These data reveal that chronic low dietary energy intake by long-term castrates, with high or low protein intake, reduces LH pulse frequency but increases the circulating levels of LH by virtue of an increase in pulse amplitude, and concomitantly increases hypothalamic NPY gene expression.

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.

IGF-I stimulation of luteinizing hormone secretion, IGF-binding proteins (IGFBPs) and expression of mRNAs for IGFs, IGF receptors and IGFBPs in the ovine pituitary gland.

Circulating concentrations of insulin-like growth factor-I (IGF-I) are reduced in juvenile sheep during nutritional growth restriction and the associated delay in puberty. Since exogenous IGF-I has been shown to stimulate luteinizing hormone (LH) secretion, it is postulated that endogenous IGF-I may act as a stimulatory metabolic signal to the pubertal ovine hypothalamo-pituitary axis, yet its site of action is unknown. Using coronal hypothalamic and pituitary sections from pubertal ewe lambs, in vitro autoradiography was used to localise 125I-labelled IGF-I binding, and gene expression for components of the IGF system was localised by in situ hybridisation using oligonucleotide probes. High concentrations of 125I-IGF-I binding were seen in the pars tuberalis (PT) and pars distalis (PD) of the pituitary, and relatively little in the hypothalamus; binding in the PT but not the PD was displaced by excess unlabelled IGF-I. Large amounts of mRNA were detected for the type-1 receptor (IGF-1R) and for IGF-binding protein (IGFBP)-5, localised to the PT and PD, and less intense specific hybridisation signals were obtained with mRNAs for IGF-II, type-2 receptor (IGF-2R) and IGFBP-3. There was some evidence for specific hybridisation to IGFBP-4 mRNA in the PT. IGF-I, IGFBP-1 and IGFBP-2 mRNAs were not detected in PT and PD. None of the genes were expressed in hypothalamic tissue. Western-ligand binding on PD extracts from male castrates revealed by their molecular weights the likely presence of IGFBPs-2, -3, and -5. Finally, cultured PD cells from abattoir-killed sheep were challenged with IGF-I (0.1, 1, 10 or 30 nM) or luteinizing hormone-releasing hormone (LHRH, 10 nM) alone, or both together. Basal LH output was stimulated by 10 nM IGF-I (120+/-11.2%, P>0.05), 30 nM IGF-I (148+/-12.8%, P<0.01), and LHRH alone (200+/-16.1%, P<0.001); there was no additive or subtractive effect of LHRH and IGF-I given together. Thus, an intrapituitary IGF system exists in sheep and the present results are consistent with an endocrine role for IGF-I in nutritional modulation of LH secretion at the level of the pituitary gland.

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.

Inhibition of luteinizing hormone secretion and expression of c-fos and corticotrophin-releasing factor genes in the paraventricular nucleus during insulin-induced hypoglycaemia in sheep.

Insulin can act within the brain to stimulate ovine luteinizing hormone (LH) secretion, but insulin-induced hypoglycaemia inhibits LH via unknown brain sites, possibly involving corticotrophin-releasing factor (CRF). Castrate male sheep, with (E+) or without (E-) subcutaneous oestradiol implants, were blood sampled every 12 min for 8 h. Insulin (0.25 or 0.5 IU/kg) was injected at 4 h via the carotid artery or jugular vein. All treatments reduced LH output with no differences between dose rate nor route of administration, but sensitivity was greater in E+ than E-sheep. There was no evidence for an effect of insulin on LH 0-1 h postinjection; however, 1-3 h after insulin, when hypoglycaemia was established, LH pulses were inhibited in both E+ and E- sheep (P<0.001). Additional intravenous (i.v.) glucose injections given 1 h (20 mmol) and 2 h (10 mmol) after insulin (0.5 IU/kg) were each followed by an LH pulse within 30 min (75% response in both E+ and E-sheep). In a separate experiment, sheep were killed 2 h after i.v. insulin (0.5 IU/kg) or saline. In-situ hybridization revealed c-fos mRNA in the paraventricular nucleus (PVN), but not in any other hypothalamic nuclei nor in the hindbrain; and this was linked with increased CRF gene expression in the PVN. Similar c-fos and CRF gene expression was seen in insulin-treated sheep given additional i.v. glucose (20 and 10 mmol, respectively, 40 and 20 min ante mortem), but not in saline-treated controls. Therefore, insulin-induced hypoglycaemia inhibited LH secretion, with oestradiol potentiating the effect, and was associated with gonadal steroid-independent c-fos gene expression and increased CRF gene expression in the PVN. The ovine PVN may be involved in mediating insulin-induced hypoglycaemic inhibition of LH by a mechanism which might involve CRF.

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.

The differential regulation of CART gene expression in a pituitary cell line and primary cell cultures of ovine pars tuberalis cells.

The cocaine-amphetamine regulated transcript (CART) encodes for a protein which has an important role in the regulation of appetite and body weight. To date, no details of the molecular events and signal transduction pathways which regulate this gene are available. We report the identification of CART gene expression in the GH3 pituitary cell line. We have used activators of the cAMP or protein kinase C (PKC) signal transduction pathways to show that, in GH3 cells, CART is transcriptionally up-regulated by activators of the cAMP signal transduction pathway. We also identify CART gene expression in ovine pars tuberalis (PT) tissue and primary cell cultures. In PT cells in contrast to GH3 cells, CART gene expression is upregulated by activators of the PKC signal transduction pathway. Cultured cells have provided a valuable resource for the detailed analysis of specific regulatory mechanisms underlying transcriptional or translational regulation of genes, signal transduction events and many other cellular processes. GH3 and PT cells may therefore provide a resource for the further detailed molecular analysis of the events regulating CART gene expression and processing.

Leptin receptor and neuropeptide Y gene expression in the sheep brain.

Leptin, a protein secretory product of adipocytes, is important in appetite control, energy balance and reproduction. In rodents, the physiological effects of leptin are centrally mediated, in part via the neuropeptide Y (NPY) system in the hypothalamus. The role of leptin in ruminants, where appropriate nutrition and reproductive status are of major economic concern, is largely unknown. To elucidate the function of leptin in sheep we have investigated putative sites of action for leptin in the brain and pituitary gland using both in-situ hybridization to detect expression of the signalling form of the leptin receptor (OB-Rb) and in-vitro autoradiography using (125I)leptin to detect sites of specific leptin binding. OB-Rb gene expression occurred in the hippocampus, cerebral cortex, preoptic area, stria terminalis and choroid plexus, and within the hypothalamus in the paraventricular (PVN), ventromedial (VHM) and arcuate (ARC) nuclei. OB-Rb gene expression in the ovine pituitary gland was not detected by in-situ hybridization. Sites of OB-Rb and NPY gene expression were compared using both in-situ hybridization on adjacent sections containing the arcuate and ventromedial nuclei, and dual in-situ hybridization on sections containing these areas. In serial sections, OB-Rb expression was found to correspond closely with that of NPY over the arcuate nuclei. Using dual in-situ hybridization, NPY expressing neurones in the arcuate nuclei were also positive for OB-Rb gene expression. Therefore, it appears that leptin may partly act via OB-Rb located on NPY neurones in the sheep hypothalamus as in the rodent.

Structure and function in the ruminant synepitheliochorial placenta: central role of the trophoblast binucleate cell in deer.

The ruminant placenta has a very uniform gross structure based on localised areas of fetomaternal membrane apposition and proliferation to form placentomes. There is no consistency, however, in the number or size of these placentomes (6-150), nor in the villus architecture of the individual placentome. The one consistent feature is the binucleate cell (BNC) population in the trophoblast. These BNC form 15-20% of the epithelium in all ruminants examined so far. They synthesise the placental lactogen hormone and other glycoproteins and migrate through tight junctions to fuse with a uterine epithelial cell to form initially fetomaternal hybrid trinucleate cells (TNC) and subsequently syncytial plaques (SP). Such SP may be transient or persist throughout pregnancy depending on the species. The wide range of deer species examined confirms the uniformity of the BNC hormone production, migration, and fusion pattern described for other ruminants. BNC migration produces predominantly transient TNC, but there are areas of SP largely restricted to the apex of the maternal crypts. Maternal large granule lymphocytes (LGL) are uniquely found in deer placentomal uterine epithelium; they are usually closely associated with TNC and SP sites, but the significance of the interactions remains to be established.