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During ovarian development stroma from the mesonephros penetrates and expands into the ovarian primordium and thus appears to be involved, at least physically, in the formation of ovigerous cords, follicles and surface epithelium. Cortical stromal development during gestation in bovine fetal ovaries (n=27) was characterised by immunohistochemistry and by mRNA analyses. Stroma was identified by immunostaining of stromal matrix collagen type I and proliferating cells were identified by Ki67 expression. The cortical and medullar volume expanded across gestation, with the rate of cortical expansion slowing over time. During gestation, the proportion of stroma in the cortex and total volume in the cortex significantly increased (P0.05). The expression levels of 12 genes out of 18 examined, including osteoglycin (OGN) and lumican (LUM), were significantly increased later in development (P

Few studies have investigated the effects of nutrition during the periconception and early gestation periods on fetal and placental development in cattle. In this study, nulliparous yearling heifers (n=360) were individually fed a diet high or low in protein (HPeri and LPeri) beginning 60 days before conception. From 24 to 98 days after conception, half of each treatment group was changed to the alternative high- or low-protein diet (HPost and LPost) yielding four groups in a 2×2 factorial design. A subset of heifers (n=46) was necropsied at 98 days after conception and fetoplacental development assessed. Placentome number and volume decreased in response to LPeri and LPost diets respectively. Absolute lung, pancreas, septum and ventricle weights decreased in LPost versus HPost fetuses, whereas the post-conception diet altered absolute and relative liver and brain weights depending on sex. Similarly, changes in fetal hepatic gene expression of factors regulating growth, glucose output and lipid metabolism were induced by protein restriction in a sex-specific manner. At term, neonatal calf and placental measures were not different. Protein restriction of heifers during the periconception and early gestation periods alters fetoplacental development and hepatic gene expression. These changes may contribute to functional consequences for progeny, but this may not be apparent from gross morphometry at birth.

Our aim was to determine the effect of exposure to maternal obesity or to maternal weight loss around conception on the programming of hepatic insulin signaling in the offspring. We used an embryo transfer model in sheep to investigate the effects of exposure to either maternal obesity or to weight loss in normal and obese mothers preceding and for 1 wk after conception on the expression of hepatic insulin-signaling and gluconeogenic factors and key miRNAs involved in insulin signaling in the offspring. We found that exposure to maternal obesity resulted in increased hepatic miR-29b (P

Heritable trait variation within a population of organisms is largely governed by DNA variations that impact gene transcription and protein function. Identifying genetic variants that affect complex functional traits is a primary aim of population genetics studies, especially in the context of human disease and agricultural production traits. The identification of alleles directly altering mRNA expression and thereby biological function is challenging due to difficulty in isolating direct effects of cis-acting genetic variations from indirect trans-acting genetic effects. Allele specific gene expression or allelic imbalance in gene expression (AI) occurring at heterozygous loci provides an opportunity to identify genes directly impacted by cis-acting genetic variants as indirect trans-acting effects equally impact the expression of both alleles. However, the identification of genes showing AI in the context of the expression of all genes remains a challenge due to a variety of technical and statistical issues. The current study focuses on the discovery of genes showing AI using single nucleotide polymorphisms as allelic reporters. By developing a computational and statistical process that addressed multiple analytical challenges, we ranked 5,809 genes for evidence of AI using RNA-Seq data derived from brown adipose tissue samples from a cohort of late gestation fetal lambs and then identified a conservative subgroup of 1,293 genes. Thus, AI was extensive, representing approximately 25% of the tested genes. Genes associated with AI were enriched for multiple Gene Ontology (GO) terms relating to lipid metabolism, mitochondrial function and the extracellular matrix. These functions suggest that cis-acting genetic variations causing AI in the population are preferentially impacting genes involved in energy homeostasis and tissue remodelling. These functions may contribute to production traits likely to be under genetic selection in the population. Refereed/Peer-reviewed

Expression of insulin-like growth factor (IGF)1 and IGF2 and their receptor (IGF1R and IGF2R) mRNA in fetal skeletal muscle are changed by variations in maternal nutrient intake. The persistence of these effects into postnatal life and their association with phenotype in beef cattle is unknown. Here we report that the cross-sectional areas of longissimus dorsi and semitendinosus (ST) muscles were greater for mature male progeny born to heifers fed low protein diets (70% vs. 240% of recommended) during the first trimester. In ST, this was accompanied by greater IGF1, IGF2 and IGF2R mRNA at 680 d. Females exposed to low protein diets during the first trimester had decreased IGF2 mRNA in ST at 680 d, however this did not result in an effect to phenotype. Exposure to low protein diets during the second trimester increased IGF1R mRNA in ST of all progeny at 680 d. Changes to expression of IGF genes in progeny skeletal muscle resulting from variations to maternal protein intake during gestation may have permanent and sex-specific effect on postnatal skeletal muscle growth. Refereed/Peer-reviewed

Maternal food restriction (FR) during pregnancy results in intrauterine growth-restricted (IUGR) offspring that show rapid catch-up growth and develop metabolic syndrome and adult obesity. However, continued nutrient restriction during nursing delays catch-up growth and prevents development of obesity. Epigenetic regulation of IGF1, which modulates growth and is synthesized and secreted by the liver, may play a role in the development of these morbidities. Control (AdLib) pregnant rats received ad libitum food through gestation and lactation, and FR dams were exposed to 50% food restriction from days 10 to 21. FR pups were nursed by either ad libitum-fed control dams (FR/AdLib) or FR dams (FR/FR). All pups were weaned to ad libitum feed. Maternal FR resulted in IUGR newborns with significantly lower liver weight and, with the use of chromatin immunoprecipitation, decreased dimethylation at H3K4 in the IGF1 region was observed. Obese adult FR/AdLib males had decreased dimethylation and increased trimethylation of H3K4 in the IGF1 region. This corresponded to an increase in mRNA expression of IGF1-A (134 ± 5%), IGF1-B (165 ± 6%), IGF1 exon 1 (149 ± 6%), and IGF1 exon 2 (146 ± 7%) in the FR/AdLib compared with the AdLib/AdLib control group. In contrast, nonobese FR/FR had significantly higher IGF1-B mRNA levels (147 ± 19%) than controls with no difference in IGF1-A, exon 1 or exon 2. Modulation of the rate of IUGR newborn catch-up growth may thus protect against IGF1 epigenetic modifications and, consequently, obesity and associated metabolic abnormalities.

There is an association between growing slowly before birth, accelerated growth in early postnatal life and the emergence of insulin resistance, visceral obesity and glucose intolerance in adult life. In this review we consider the pathway through which intrauterine growth restriction (IUGR) leads to the initial increase in insulin sensitivity and to catch-up growth. We also discuss the importance of the early insulin environment in determining later visceral adiposity and the intrahepatic mechanisms that may result in the emergence of glucose intolerance in a subset of IUGR infants. We present evidence that a key fetal adaptation to poor fetal nutrition is an upregulation of the abundance of the insulin receptor in the absence of an upregulation of insulin signalling in fetal skeletal muscle. After birth, however, there is an upregulation in the abundance of the insulin receptor and the insulin signalling pathway in the IUGR offspring. Thus, the origins of the accelerated postnatal growth rate experienced by IUGR infants lie in the fetal adaptations to a poor nutrient supply. We also discuss how the intracellular availability of free fatty acids and glucose within the visceral adipocyte and hepatocyte in fetal and neonatal life are critical in determining the subsequent metabolic phenotype of the IUGR offspring. It is clear that a better understanding of the relative contributions of the fetal and neonatal nutrient environment to the regulation of key insulin signalling pathways in muscle, visceral adipose tissue and the liver is required to support the development of evidence-based intervention strategies and better outcomes for the IUGR infant. Refereed/Peer-reviewed

The fetus makes a number of physiological adaptations to a restriction of placental substrate supply, including a decrease in body growth and an increase in peripheral vasoconstriction which maintains mean arterial pressure (MAP) and supports a redistribution of cardiac output to key fetal organs. It is not known, however, whether chronic restriction of placental substrate supply results in an enhanced or diminished role for vasodilators such as endothelial nitric oxide in the regulation of MAP. We hypothesised that there is an increased contribution of NO to blood pressure regulation in growth restricted fetuses and that a 2 h infusion of a nitric oxide synthase inhibitor, N(omega)-nitro-L-arginine methyl ester (L-NAME) would result in an augmented rise in MAP in chronically hypoxemic, placentally restricted (PR, n ¼ 8) fetuses compared to controls (n ¼ 6) in late gestation. There was no difference in the increase in fetal MAP and decrease in HR during L-NAME infusion between Control and PR fetuses. In the PR group, fetuses with lower mean gestational PaO2 had a lower increase in MAP during L-NAME infusion. Thus we have found no evidence for an enhanced role of NO in the maintenance of MAP in the chronically hypoxemic IUGR fetus. Refereed/Peer-reviewed

Epidemiological studies have shown that infants exposed to an increased supply of nutrients before birth are at increased risk of type 2 diabetes in later life. We have investigated the hypothesis that fetal overnutrition results in reduced expression and phosphorylation of the cellular fuel sensor, AMP-activated kinase (AMPK) in liver and skeletal muscle before and after birth. From 115 days gestation, ewes were fed either at or approximately 55% above maintenance energy requirements. Postmortem was performed on lamb fetuses at 139-141 days gestation (n = 14) and lambs at 30 days of postnatal age (n = 21), and liver and quadriceps muscle were collected at each time point. The expression of AMPKalpha1 and AMPKalpha2 mRNA was determined by quantitative RT-PCR (qRT-PCR). The abundance of AMPKalpha and phospho-AMPKalpha (P-AMPKalpha) was determined by Western blot analysis, and the proportion of the total AMPKalpha pool that was phosphorylated in each sample (%P-AMPKalpha) was determined. The ratio of AMPKalpha2 to AMPKalpha1 mRNA expression was lower in fetuses compared with lambs in both liver and muscle, independent of maternal nutrition. Hepatic %P-AMPKalpha was lower in both fetuses and lambs in the Overfed group and %P-AMPKalpha in the lamb liver was inversely related to plasma glucose concentrations in the first 24 h after birth (r = 0.73, P0.025). There was no effect of maternal overnutrition on total AMPKalpha or P-AMPKalpha abundance in liver or skeletal muscle. We have, therefore, demonstrated that AMPKalpha responds to signals of increased nutrient availability in the fetal liver. Suppression of hepatic AMPK phosphorylation may contribute to increased glucose production, and basal hyperglycemia, present in lambs of overfed ewes in early postnatal life.

Placental restriction and insufficiency are associated with altered patterns of placental growth, morphology, substrate transport capacity, growth factor expression, and glucocorticoid exposure. We have used a pregnant sheep model in which the intrauterine environment has been perturbed by uterine carunclectomy (Cx). This procedure results in early restriction of placental growth and either the development of chronic fetal hypoxemia (PaO(2 17 mmHg). Based on fetal PaO2, Cx, and Control ewes were assigned to either a normoxemic fetal group (Nx) or a hypoxemic fetal group (Hx) in late gestation, resulting in 4 groups. Cx resulted in a decrease in the volumes of fetal and maternal connective tissues in the placenta and increased placental mRNA expression of IGF2, vascular endothelial growth factor (VEGF), VEGFR-2, ANGPT2, and TIE2. There were reduced volumes of trophoblast, maternal epithelium, and maternal connective tissues in the placenta and a decrease in placental GLUT1 and 11 beta HSD2 mRNA expression in the Hx compared to Nx groups. Our data show that early restriction of placental growth has effects on morphological and functional characteristics of the placenta in late gestation, independent of whether the fetus becomes hypoxemic. Similarly, there is a distinct set of placental changes that are only present in fetuses that were hypoxemic in late gestation, independent of whether Cx occurred. Thus, we provide further understanding of the different placental cellular and molecular mechanisms that are present in early placental restriction and in the emergence of later placental insufficiency. Refereed/Peer-reviewed

The present study tested the hypothesis that exposure to an increased level of maternal nutrition before birth results in altered expression of adipogenic, lipogenic, and adipokine genes in adipose tissue in early postnatal life. Pregnant ewes were fed either at or approximately 50% above maintenance energy requirements during late pregnancy, and quantitative RT-PCR was used to measure peroxisome proliferator-activated receptor (PPAR)-gamma, lipoprotein lipase (LPL), glycerol-3-phosphate-dehydrogenase (G3PDH), adiponectin, and leptin mRNA expression in perirenal (PAT) and sc adipose tissue (SCAT) in the offspring on postnatal d 30. Relative SCAT mass was higher in lambs of well-fed ewes (40.0 +/- 4.0 vs. 22.8 +/- 3.3 g/kg, P < 0.05) and was directly related to plasma insulin in the first 24 h after birth and to G3PDH and LPL expression. The expression of leptin mRNA in both the SCAT and PAT depots was higher (P < 0.05) in lambs of well-fed ewes. PPARgamma adiponectin, LPL, and G3PDH mRNA expression were not, however, different between well-fed and control groups in either depot. Relative PPARgamma expression in SCAT was directly related to plasma insulin concentrations in the first 24 h after birth (r(2) = 0.23; P < 0.05), and G3PDH and LPL expressions were also positively correlated with PPARgamma expression (r(2) = 0.27; P < 0.05). We have demonstrated that exposure to increased prenatal nutrition increases leptin expression at 1 month of age in both PAT and SCAT. The results of this study provide evidence that the nutritional environment before and immediately after birth can influence the development of adipose tissue in early postnatal life.

We investigated the effects of an intrafetal infusion of IGF-I on adrenal growth and expression of the adrenal steroidogenic and catecholamine-synthetic enzyme mRNAs in the sheep fetus during late gestation. Fetal sheep were infused for 10 d with either IGF-I (26 μg/kg·h; n = 14) or saline (n = 10) between 120 and 130 d gestation, and adrenal glands were collected for morphological analysis and determination of the mRNA expression of steroidogenic and catecholamine-synthetic enzymes. Fetal body weight was not altered by IGF-I infusion; however, adrenal weight was significantly increased by 145% after IGF-I infusion. The density of cell nuclei within the fetal adrenal cortex (the zona glomerulosa and zona fasciculata), and within the adrenaline synthesizing zone of the adrenal medulla, was significantly less in the IGF-I-infused fetuses compared with the saline-infused group. Thus, based on cell-density measurements, there was a significant increase in cell size in the zona glomerulosa and zona fasciculata of the adrenal cortex and in the adrenaline-synthesizing zone of the adrenal medulla. There was no effect of IGF-I infusion on the adrenal mRNA expression of the steroidogenic or catecholamine-synthetic enzymes or on fetal plasma cortisol concentrations. In summary, infusion of IGF-I in late gestation resulted in a marked hypertrophy of the steroidogenic and adrenaline-containing cells of the fetal adrenal in the absence of changes in the mRNA levels of adrenal steroidogenic or catecholamine-synthetic enzymes or in fetal plasma cortisol concentrations. Thus, IGF-I infusion results in a dissociation of adrenal growth and function during late gestation.

Placental insufficiency, resulting in restriction of fetal substrate supply, is a major cause of intrauterine growth restriction (IUGR) and increased neonatal morbidity. Fetal adaptations to placental restriction maintain the growth of key organs, including the heart, but the impact of these adaptations on individual cardiomyocytes is unknown. Placental and hence fetal growth restriction was induced in fetal sheep by removing the majority of caruncles in the ewe before mating (placental restriction, PR). Vascular surgery was performed on 13 control and 11 PR fetuses at 110–125 days of gestation (term: 150 ± 3 days). PR fetuses with a mean gestational Po2 < 17 mmHg were defined as hypoxic. At postmortem (135 days), fetal hearts were collected, and cardiomyocytes were isolated and fixed. Proliferating cardiomyocytes were counted by immunohistochemistry of Ki67 protein. Cardiomyocytes were stained with methylene blue to visualize the nuclei, and the proportion of mononucleated cells and length and width of cardiomyocytes were measured. PR resulted in chronic fetal hypoxia, IUGR, and elevated plasma cortisol concentrations. Although there was no difference in relative heart weights between control and PR fetuses, there was an increase in the proportion of mononucleated cardiomyocytes in PR fetuses. Whereas mononucleated and binucleated cardiomyocytes were smaller, the relative size of cardiomyocytes when expressed relative to heart weight was larger in PR compared with control fetuses. The increase in the relative proportion of mononucleated cardiomyocytes and the relative sparing of the growth of individual cardiomyocytes in the growth-restricted fetus are adaptations that may have long-term consequences for heart development in postnatal life.

The fetal pituitary-adrenal axis plays a key role in the fetal response to intrauterine stress and in the timing of parturition. The fetal sheep adrenal gland is relatively refractory to stimulation in midgestation (90–120 days) before the prepartum activation, which occurs around 135 days gestation (term = 147 ± 3 days). The mechanisms underlying the switch from adrenal quiescence to activation are unclear. Therefore, we have investigated the expression of suppressor of cytokine signaling-3 (SOCS-3), a putative inhibitor of tissue growth in the fetal sheep adrenal between 50 and 145 days gestation and in the adrenal of the growth-restricted fetal sheep in late gestation. SOCS-3 is activated by a range of cytokines, including prolactin (PRL), and we have, therefore, determined whether PRL administered in vivo or in vitro stimulates SOCS-3 mRNA expression in the fetal adrenal in late gestation. There was a decrease ( P < 0.005) in SOCS-3 expression in the fetal adrenal between 54 and 133 days and between 141 and 144 days gestation. Infusion of the dopaminergic agonist, bromocriptine, which suppressed fetal PRL concentrations but did not decrease adrenal SOCS-3 mRNA expression. PRL administration, however, significantly increased adrenal SOCS-3 mRNA expression ( P < 0.05). Similarly, there was an increase ( P < 0.05) in SOCS-3 mRNA expression in adrenocortical cells in vitro after exposure to PRL (50 ng/ml). Placental and fetal growth restriction had no effect on SOCS-3 expression in the adrenal during late gestation. In summary, the decrease in the expression of the inhibitor SOCS-3 after 133 days gestation may be permissive for a subsequent increase in fetal adrenal growth before birth. We conclude that factors other than PRL act to maintain adrenal SOCS-3 mRNA expression before 133 days gestation but that acute elevations of PRL can act to upregulate adrenal SOCS-3 expression in the sheep fetus during late gestation.

A series of epidemiological, clinical and experimental studies have shown that there are associations between the fetal and neonatal nutritional environment and the amount and distribution of adipose tissue in adult life. This review considers the evidence for these relationships and discusses the potential impact of the prenatal nutritional experience on the development of the endocrine and neuroendocrine systems that regulate energy balance, with a particular emphasis on the role of the adipocyte-derived hormone, leptin. In the rodent, leptin derived from the mother may exert an important influence on the development of the appetite regulatory neural network and on the subsequent regulation of leptin synthesis and the risk for obesity in the offspring. In species such as the human and sheep, there is also recent evidence that the synthesis and secretion of adipocyte-derived hormones, such as leptin, are regulated in fetal life. Furthermore, the hypothalamic neuropeptides that regulate energy intake and expenditure in adult life are also present within the fetal brain and may be regulated by the prevailing level of maternal and hence fetal nutrient and hormonal signals, including leptin. This work is important in determining those initiating mechanisms within the ‘fat–brain’ axis in early life that precede the development of adult obesity.

Depression during pregnancy is frequently treated with the selective serotonin reuptake inhibitor (SSRI), fluoxetine (FX), commonly known as Prozac (Eli Lilly & Co, Indianapolis, IN). FX potentiates serotoninergic neurotransmission and serotonin has been implicated in the regulation of circadian rhythms. We have therefore investigated the effect of chronic administration of FX on maternal and fetal circadian rhythms in sheep. Following an initial bolus dose of 70 mg FX, an 8-day continuous infusion of FX (n = 11, 98.5 μg/kg • d) was performed. Controls (n = 13) were treated with sterile water vehicle only. Maternal and fetal plasma melatonin and prolactin concentrations were determined every 3 hours for 24 hours and then every 6 hours for 24 hours beginning on the fourth day of infusion. FX treatment did not alter either the basal or circadian rhythms of either maternal or fetal plasma melatonin and prolactin concentrations. Fetal cardiovascular and behavioral state parameters were measured continuously. While the incidence of low-voltage (LV) electrocortical (ECOG) activity was significantly reduced in fetuses in the FX group, there was no effect of FX on the diurnal rhythms in fetal arterial pressure, heart rate, breathing movements, or behavioral state. These results show that maternal FX treatment does not result in significant alterations in maternal and fetal hormonal and behavioral circadian rhythms.

It has been proposed that maternal nutrient restriction may alter the functional development of the adipocyte and the synthesis and secretion of the adipocyte-derived hormone, leptin, before birth. We have investigated the effects of restricted periconceptional undernutrition and/or restricted gestational nutrition on fetal plasma leptin concentrations and fetal adiposity in late gestation. There was no effect of either restricted periconceptional or gestational nutrition on maternal or fetal plasma leptin concentrations in singleton or twin pregnancies during late gestation. In ewes carrying twins, but not singletons, maternal plasma leptin concentrations in late gestation were directly related to the change in ewe weight that occurred during the 60 days before mating [maternal leptin = 0.9 (change in ewe weight) + 7.8; r = 0.6, P < 0.05]. In twin, but not singleton, pregnancies, there was also a significant relationship between maternal and fetal leptin concentrations (maternal leptin = 0.5 fetal leptin + 4.2, r = 0.63, P < 0.005). The relative mass of perirenal fat was also significantly increased in twin fetal sheep in the control-restricted group (6.0 ± 0.5) compared with the other nutritional groups (control-control: 4.1 ± 0.4; restricted-restricted: 4.4 ± 0.4; restricted-control: 4.3 ± 0.3). In conclusion, the impact of maternal undernutrition on maternal plasma leptin concentrations during late gestation is dependent on fetal number. Furthermore, we have found that there is an increased fetal adiposity in the twins of ewes that experienced restricted nutrition throughout gestation, and this may be important in the programming of postnatal adiposity.

A range of epidemiological and experimental studies have indicated that suboptimal nutrition at different stages of gestation is associated with an increased prevalence of adult hypertension, cardiovascular disease, and obesity. The timing of prenatal nutrient restriction is important in determining postnatal outcomes-including obesity. The present study, aimed to determine the extent to which fetal adiposity and expression of the key thermogenic protein, uncoupling protein (UCP)1, are altered by restriction of maternal nutrient intake imposed during four different periods, starting from before conception. Maternal nutrient intake was restricted from 60 days before until 8 days after mating (periconceptional nutrient restriction; R-C), from 60 days before mating and throughout gestation (R-R), from 8 days gestation until term (C-R), or from 115 days gestation until term. Fetal perirenal adipose tissue (PAT) was sampled near to term at approximately 143 days. UCP1 mRNA, but not protein, abundance in PAT was increased in fetuses in the R-R group (C-C 63 +/- 18; R-C 83 +/- 43; C-R 103 +/- 38; R-R 167 +/- 50 arbitrary units (P < 0.05)). In contrast, the abundance of UCP1 mRNA, but not protein, in fetal PAT was decreased when maternal nutrition was restricted from 115 days gestation. The major effect of maternal nutrient restriction on adipose tissue deposition occurred in the C-R group, in which the proportion of fetal fat was doubled, whereas maternal nutrient restriction from 115 days gestation reduced fetal fat deposition. In conclusion, there are differential effects of maternal and therefore fetal nutrient restriction on UCP1 mRNA expression and fetal fat mass and these effects are dependent on the timing and duration of nutrient restriction.

We have investigated the effects of a 5 day infusion of cortisol into fetal sheep, in which the hypothalamus and pituitary were surgically disconnected (HPD), on fetal pituitary-adrenal function. Fetal HPD and vascular catheterization were carried out at between 104 and 124 days gestation. Cortisol was administered (3.5 mg 24 h-1) for 120 h between 134 and 140 days (HPD + F group; n = 5) and saline was administered during the same gestational age range to HPD (HPD group; n = 12) and intact fetal sheep (Intact group; n = 6). Cortisol infusion into the HPD fetal sheep did not suppress the mRNA levels for Proopiomelanocortin (POMC) in the fetal anterior pituitary at 139/140 days gestation (POMC mRNA: 18S rRNA: Intact 0.40 +/- 0.05; HPD 0.56 +/- 0.07; HPD + F 0.49 +/- 0.07). Similarly, there was no significant effect of either HPD or cortisol infusion on the plasma concentrations of immunoreactive (ir) ACTH or ACTH(1-39). The adrenal: fetal body weight ratio was significantly higher, however, in the HPD + F (88.4 +/- 8.7 mg kg-1) and Intact groups (84.1 +/- 5.6 mg kg-1) when compared with the HPD fetal sheep (63.7 +/- 5.4 mg kg-1). The ratio of total IGF-II mRNA: 18S rRNA was similar in the adrenals of the Intact (0.48 +/- 0.09), HPD (0.78 +/- 0.09) and HPD + F (0.71 +/- 0.11) groups. The ratios of CYPIIA1, 3 beta-HSD and CYP21A1 mRNA: 18S rRNA were significantly lower in adrenals from the HPD group when compared to those in the Intact group and were not restored to normal by cortisol infusion. We have therefore demonstrated that cortisol does not act directly at the fetal pituitary to suppress POMC synthesis or ACTH secretion in late gestation. Cortisol does, however, stimulate fetal adrenal growth after HPD in the absence of any effects on adrenal IGF-II or steroidogenic enzyme mRNA levels. The data provide evidence that an intact hypothalamic-pituitary axis and cortisol each play an important role in the stimulation of adrenal growth and steroidogenesis which occurs during the last 10-15 days of gestation in the sheep.

The placenta is the primary interface between the fetus and mother and plays an important role in maintaining fetal development and growth by facilitating the transfer of substrates and participating in modulating the maternal immune response to prevent immunological rejection of the conceptus. The major substrates required for fetal growth include oxygen, glucose, amino acids and fatty acids, and their transport processes depend on morphological characteristics of the placenta, such as placental size, morphology, blood flow and vascularity. Other factors including insulin-like growth factors, apoptosis, autophagy and glucocorticoid exposure also affect placental growth and substrate transport capacity. Intrauterine growth restriction (IUGR) is often a consequence of insufficiency, and is associated with a high incidence of perinatal morbidity and mortality, as well as increased risk of cardiovascular and metabolic diseases in later life. Several different experimental methods have been used to induce placental insufficiency and IUGR in animal models and a range of factors that regulate placental growth and substrate transport capacity have been demonstrated. While no model system completely recapitulates human IUGR, these animal models allow us to carefully dissect cellular and molecular mechanisms to improve our understanding and facilitate development of therapeutic interventions. Refereed/Peer-reviewed

Periconceptional nutrition (PCN) can influence foetal hypothalamo-pituitary adrenal (HPA) axis function and alter cortisol secretion with possible consequences for maturation and growth of major organs, gestation length and behaviour. We examined effects of PCN on phenotype and survival of the neonatal lamb in 466 Merino ewes allocated to treatments providing 70%, 100% and 150% respectively, of maintenance requirements for 17 days prior and 6 days after insemination. Gestation length and birth weight for lambs in PCN treatment groups was similar (P > 0.05) but low PCN decreased the size of the neonate (crown-rump-length and metacarpal length P < 0.05). A subset of lambs euthanased at 5 days of age further showed that low PCN decreased the amount of peri-renal fat (P < 0.05) and increased liver mass (P < 0.05) while high PCN increased neck thymus and ovary mass (P < 0.05). Neonatal lambs from low PCN ewes returned faster to their mothers after release (P < 0.05) and contacted the udder in the shortest time (P < 0.05). Significant interactions between PCN treatment and sex (P < 0.05) and between PCN treatment and ewe age (P < 0.05) were also observed for time lambs took to follow the ewe. Survival of lambs was similar but potential differences may have been masked by favourable weather conditions. In conclusion, this study provides evidence of significant changes in lamb growth and development dependent on PCN and, for the first time, links these changes with significant changes in behaviour of the neonate. The impact of these effects on lamb survival and potential reproductive capacity of female offspring remains to be determined.

Maternal obesity is associated with an increased risk of developing gestational diabetes mellitus and it also results in an increased risk of giving birth to a large baby with increased fat mass. Furthermore, it is also contributes to an increased risk of obesity and insulin resistance in the offspring in childhood, adolescence and adult life. It has been proposed that exposure to maternal obesity may therefore result in an ‘intergenerational cycle’ of obesity and insulin resistance. There is significant interest in whether exposure to maternal obesity around the time of conception alone contributes directly to poor metabolic outcomes in the offspring and whether dieting in the obese mother before pregnancy or around the time of conception has metabolic benefits for the offspring. This review focusses on experimental and clinical studies that have investigated the specific impact of exposure to maternal obesity during the periconceptional period alone or extending beyond conception on adipogenesis, lipogenesis and on insulin signalling pathways in the fat, liver and muscle of the offspring. Findings from these studies highlight the need for a better evidence base for the development of dietary interventions in obese women before pregnancy and around the time of conception to maximize the metabolic benefits and minimize the metabolic costs for the next generation. Refereed/Peer-reviewed

A range of pathophysiological factors can result in a perturbation or restriction of fetal growth, and the cardiovascular, neuroendocrine and metabolic adaptations of the fetus to these stimuli will depend on their nature, timing and intensity. The critical importance of these physiological adaptations for both immediate survival and long-term health outcomes has provided an impetus for experimental studies of the nature and consequences of specific fetal adaptations to a poor intrauterine environment. This review summarizes data from recent studies that have focused on the responses of the fetal cardiovascular, sympathoadrenal, hypothalamo-pituitary-adrenal and renin-angiotensin systems to experimental restriction of placental function in the sheep and discusses the consequences of these adaptations for fetal, neonatal and adult health.

This article reviews the fetal endocrine system in sheep, a species that has a long gestation and primarily produces a singleton fetus. Attention is focused on information that is applicable to humans. The endocrinology of metabolic homeostasis in sheep fetuses is well adapted to respond to a range of metabolic challenges, including placental restriction and maternal undernutrition. A small placenta results in hypoxaemia, hypoglycaemia, reduced abundance of anabolic hormones, and fetal growth restriction. Fetuses with restricted growth are characterized by tissue-specific reductions in hormone receptor mRNA, for example mRNA for the long form of prolactin receptor is reduced in adipose tissue. In contrast, the adipose tissue of fetuses with accelerated growth, stimulated by increasing maternal nutrition in the second half of gestation, has more protein for the long form of the prolactin receptor and more uncoupling protein 1, by which large amounts of heat are generated at birth. Maternal undernutrition in early gestation, coinciding with the period of rapid placental growth, initially restricts placental growth, but when mothers are fed to requirements, a longer fetus results with a disproportionately large placenta. This nutritional manipulation replicates, in part, epidemiological findings from the Dutch famine of 1944-1945, for which the offspring are at increased risk of adult obesity.

In the sheep there is a rapid increase in fetal adrenal growth and steroidogenesis during the last 10-15 days gestation (term = 147+/-3 days gestation). In the rat, peptides derived from the N-terminal region of POMC play a role in compensatory adrenal growth and in potentiation of ACTH-induced steroidogenesis. We therefore investigated the effects of infusion of bovine N-POMC-(1-77) and its biosynthetic derivative, N-POMC-(1-49) on adrenal growth and on the expression of adrenal steroidogenic enzymes in the late gestation sheep fetus. Twenty-seven pregnant ewes were used in this study. Fetal vascular catheters were inserted between 116-125 days gestation, and purified bovine N-POMC-(1-77) (2 microg/ml x h), N-POMC-(1-49) (2 microg/ml x h) and saline were each infused for 48 h between 136 and 138 days gestation. Intrafetal infusion of N-POMC-(1-77) resulted in an increased adrenal/fetal body weight ratio (94.6+/-5.7 mg/kg) compared with that in saline-infused (75.6+/-1.8 mg/kg), but not N-POMC-(1-49)-infused (82.7+/-6.1 mg/kg), fetal sheep. The ratio of CYP17 messenger RNA (mRNA) to 18S ribosomal RNA was also significantly higher in fetal adrenals ofthe N-POMC-(1-77)-infused group (49.1+/-4.7) compared with that in either the N-POMC-(1-49)-infused (20.4+/-6.4) or saline-infused (15.2+/-4.4) group. There was no difference, however, in the ratios of adrenal CYP11A1 mRNA/3beta-hydroxysteroid dehydrogenase/delta5,delta4-isomerase mRNA and CYP21A1 mRNA/18S ribosomal RNA among the N-POMC-(1-77)-, N-POMC-(1-49)-, and saline-infused groups. There was also no significant change in either plasma cortisol or ACTH concentrations in response to the infusion of either N-POMC-(1-77) or N-POMC-(1-49). In summary, intrafetal infusion of N-POMC-(1-77) stimulated fetal adrenal growth and resulted in a specific increase in adrenal CYP17 gene expression in late gestation. N-POMC-(1-77) may therefore play a modulatory role in the increase in fetal adrenal growth and steroidogenesis that occurs before birth.

Leptin mRNA was measured in adipose tissue of fetal sheep by reverse transcription polymerase chain reaction (RTPCR). Abundance of leptin mRNA relative to beta-actin mRNA in fetal perirenal adipose tissue increased (P

We have investigated the separate actions of hypothalamo-pituitary disconnection (HPD), with or without cortisol administration, and changes in the external photoperiod on the regulation of the levels of messenger RNA (mRNA) encoding long (PRLR1) and short (PRLR2) forms of PRL receptor in the liver of the fetal lamb. In pregnant Merino ewes (n = 20), the hypothalamus and pituitary were surgically disconnected in 13 fetuses (HPD group), and fetal vascular catheters were implanted in the HPD group and in an additional 7 fetuses (intact + saline group) between 104-120 days gestation (d). Fetal sheep in the HPD group were infused with either cortisol (3.5 mg/4.8 ml saline/24 h; HPD + F; n = 5) or saline for 5 days between 134-141 d, and saline was also infused in the intact group within the same gestational age range. A second group of pregnant ewes (n = 12) was kept in a 12-h light, 12-h dark cycle from 70 d until implantation of fetal vascular catheters between 106-120 d, after which ewes were allocated to either a long photoperiod (16 h of light, 8 h of darkness; LL group; n = 6) or a short photoperiod (8 h of light, 16 h of darkness; SL group; n = 6) regimen. Circulating cortisol concentrations were higher (P0.05) in the intact fetal sheep (18.7 +/- 3.8 nmol/liter) than in the HPD + saline group (1.5 +/- 0.6 nmol/liter), and were further increased (P0.05) in the HPD + cortisol group (97.4 +/- 23.7 nmol/liter). Fetal PRL concentrations were lower (P0.05) in the HPD + saline (10.6 +/- 4.3 ng/ml) and HPD + cortisol (5.6 +/- 2 ng/ml) groups compared with those in the intact group (38.9 +/- 6.8 ng/ml). The levels of hepatic PRLR mRNA were higher (P0.05) in the intact (PRLR1, 27.4 +/- 6.1; PRLR2, 17.7 +/- 2.5) and HPD + cortisol (PRLR1, 23.4 +/- 0.4; PRLR2, 15.3 +/- 3.0) groups than in the HPD + saline group (PRLR1, 10.6 +/- 1.8; PRLR2, 8.9 +/- 1.8) at 140/141 d. The mean plasma PRL concentration in the LL group (70 +/- 9 ng/ml) was higher (P0.05) than that in the SL group (34 +/- 15 ng/ml), whereas the levels of hepatic PRLR1 mRNA (LL group, 4.6 +/- 0.9; SL group, 4.3 +/- 0.8) and PRLR2 mRNA (LL group, 3.4 +/- 0.4; SL group, 3.0 +/- 0.5) at 140-141 d were not different. These data indicate that cortisol acts directly or indirectly to maintain hepatic PRLR mRNA levels in the sheep fetus during late pregnancy. In contrast, changes in the external photoperiod and circulating PRL concentrations in the sheep fetus do not directly alter PRLR expression in the fetal liver. These studies provide further insight into the role that the PRL axis may play in the transduction of signals about the external environment to the fetus as it prepares for the transition to extrauterine life.

1. The ability of the fetal adrenal medulla to respond directly to hypoxaemia and secrete catecholamines before the development of a functional innervation of the gland is essential for intrauterine survival. The cellular mechanisms involved in this response to low PO2 are not known, although the presence of oxygen-sensitive K+ channels in carotid body chemoreceptor cells and other sites suggests that these might underlie the chromaffin cell response. 2. Whole-cell patch-clamp techniques have been used to study K+ currents during normoxia and hypoxia in chromaffin cells isolated from the adrenal glands of fetal sheep. 3. Two types of chromaffin cells were observed, those with a fast inactivating K+ current and a larger capacitance and those with a delayed K+ current and smaller capacitance. No cell showed both types of current. The fast inactivating current showed voltage-dependent inactivation and was blocked by 1 mM 4-aminopyridine, characteristics of an IA-type current. The delayed current had two components, a TEA-sensitive, Ca2+-dependent current and a component with the kinetic behaviour of a delayed rectifier. 4. Both types of current were oxygen sensitive. The IA-type current was reduced by 27.4 +/- 3.2 % when the PO2 was reduced to about 15 mmHg. With the delayed current, hypoxia reduced the amplitude by 26.9 +/- 2.4 %, largely by reduction of the Ca2+-dependent component. 5. In the presence of hypoxia, reduction in the amplitude of these oxygen-sensitive K+ currents would increase the frequency and duration of action potentials, leading to increased activation of the L-type Ca2+ channels, influx of Ca2+ and the subsequent secretion of catecholamines.

We investigated whether the fetal lamb can construct a photoperiodic history in utero. We measured the fetal PRL response to a 12-h photoperiod in intact fetal sheep and in fetal sheep after hypothalamo-pituitary disconnection (HPD), following exposure of the ewe to either a long (16 h L) or short (8 h L) photoperiod for 50 days in early pregnancy. Ewes were maintained on either a long light (LL, n = 20) or a short light (SL, n = 19) regimen from 57 days gestation until fetal HPD (pre-LL, n = 7; pre-SL, n = 7) or sham surgery (pre-LL, n = 13; pre-SL, n = 12) was performed at 99-113 days gestation. All ewes were housed in a 12-h photoperiod from surgery until 140 days gestation. In HPD fetal sheep previously exposed to SL, fetal PRL concentrations were significantly higher (P0.05) after 20 days in the 12-h L regimen than previously (0-5 days, 3.2 +/- 0.6 ng/ml; 21-25 days, 5.6 +/- 1.4 ng/ml). In the HPD fetal sheep previously exposed to LL, however, fetal PRL concentrations significantly decreased (P0.05) after 5 days exposure to the 12-h L regimen (6.7 +/- 2.9 ng/ml) and remained low throughout the remaining study period (31-35 days, 1.7 +/- 0.5 ng/ml). In contrast, in the sham group there was no effect of photoperiodic history on the gestational age profile of fetal PRL, and PRL concentrations increased significantly (F = 22.4, P0.001) in fetal sheep previously exposed to either SL or LL. Fetal PRL concentrations were significantly higher (P0.05) after 121 days gestation in the 12-h L regimen in all sham fetal sheep (110 days, pre-SL 6.4 +/- 0.3 ng/ml, pre-LL 12.0 +/- 3.3 ng/ml; 121-125 days, pre-SL 20.0 +/- 3.9 ng/ml, pre-LL 25.9 +/- 4.4 ng/ml). TRH (50 microg) was administered i.v. to all fetal sheep at 130-134 days gestation. There was a significant fetal PRL response to TRH in both the HPD (F = 20.9, P0.001) and sham (F = 31.3, P0.001) groups. There was no difference, however, in the PRL response to TRH in fetal sheep previously exposed to SL or LL in either the HPD or sham groups. The maximum percentage changes in PRL occurred at +10 min after TRH administration in the HPD (pre-SL, 421 +/- 75%; pre-LL, 555 +/- 76%) and sham groups (pre-SL, 394 +/- 68%; pre-LL, 369 +/- 59%). In summary, therefore, we have demonstrated that there is an effect of photoperiodic history on the PRL response to an intermediate photoperiod in utero in HPD fetal sheep. It appears, however, that the effect of photoperiodic history on PRL secretion in intact fetal sheep is either masked or suppressed by the stimulatory effect of factors associated with an increase in gestational age acting at the fetal hypothalamus.

We have investigated the effect of increasing gestational age and cortisol on prolactin receptor (PRLR) gene expression in the fetal sheep liver during late gestation. RNA was extracted from the liver of sheep fetuses between 90 and 144 days (d) gestation (n = 18) and after intrafetal infusion of either cortisol (2-2.5 mg cortisol i.v./24 h; n = 6) or saline (n = 6) between 109 and 116 d gestation. A ribonuclease protection assay for the mRNAs encoding the long (PRLR1) and short (PRLR2) forms of the PRLR was developed using an antisense RNA probe complementary to ovine PRLR2. There was a significant increase (p < 0.05) in the relative levels of liver PRLR1: GAPDH mRNA and PRLR2: GAPDH mRNA levels in fetal sheep between 90 and 144d gestation (PRLR1 mRNA: 90-95 d 0.6 +/- 0.1, 131-133 d 1.2 +/- 0.2, 141-144 d 3.6 +/- 0.5; PRLR2 mRNA: 90-95 d 0.7 +/- 0.1; 131-133 d 1.4 +/- 0.2, 141-144 d 3.0 +/- 0.4). The relative levels of liver PRLR1 and PRLR2: GAPDH mRNA levels were higher (p < 0.05) after cortisol administration (1.7 +/- 0.3 and 0.9 +/- 0.1 respectively) when compared with the saline infused group (0.7 +/- 0.1 and 0.5 +/- 0.1 respectively). We have demonstrated therefore that there is in increase in the levels of the mRNA encoding PRLR1 and PRLR2 in the fetal sheep liver during late gestation and that physiological increases in fetal cortisol stimulate PRLR1 and PRLR2 expression in the liver of the sheep fetus. These data suggest that fetal PRL may play a role in the growth and maturation of the fetal liver which occurs before birth.

Exposure to maternal obesity before and/or throughout pregnancy may increase the risk of obesity and insulin resistance in the offspring in childhood and adult life, therefore, resulting in its transmission into subsequent generations. We have previously shown that exposure to maternal obesity around the time of conception alone resulted in increased adiposity in female lambs. Changes in the abundance of insulin signalling molecules in skeletal muscle and adipose tissue precede the development of insulin resistance and type 2 diabetes. It is not clear, however, whether exposure to maternal obesity results in insulin resistance in her offspring as a consequence of the impact of increased adiposity on skeletal muscle or as a consequence of the programming of specific changes in the abundance of insulin signalling molecules in this tissue. We have used an embryo transfer model in the sheep to investigate the effects of exposure to either maternal obesity or to weight loss in normal and obese mothers preceding and for one week after conception on the expression and abundance of insulin signalling molecules in muscle in the offspring. We found that exposure to maternal obesity resulted in lower muscle GLUT-4 and Ser 9 phospho-GSK3α and higher muscle GSK3α abundance in lambs when compared to lambs conceived in normally nourished ewes. Exposure to maternal weight loss in normal or obese mothers, however, resulted in lower muscle IRS1, PI3K, p110β, aPKCζ, Thr 642 phospho-AS160 and GLUT-4 abundance in the offspring. In conclusion, maternal obesity or weight loss around conception have each programmed specific changes on subsets of molecules in the insulin signalling, glucose transport and glycogen synthesis pathways in offspring. There is a need for a stronger evidence base to ensure that weight loss regimes in obese women seeking to become pregnant minimize the metabolic costs for the next generation.

Exposure to dietary restriction during the periconceptional period in either normal or obese ewes results in increased adrenal growth and a greater cortisol response to stress in the offspring, but the mechanisms that programme these changes are not fully understood. Activation of the angiotensin type 1 receptor (AT1R) has been demonstrated to stimulate adrenal growth and steroidogenesis. We have used an embryo transfer model in the sheep to investigate the effects of exposure to dietary restriction in normal or obese mothers from before and 1 week after conception on the methylation status, expression, abundance and localisation of key components of the renin–angiotensin system (RAS) in the adrenal of post-natal lambs. Maternal dietary restriction in normal or obese ewes during the periconceptional period resulted in an increase in angiotensin-converting enzyme (ACE) and AT1R abundance in the absence of changes in the methylation status or mRNA expression ofACEandAT1Rin the adrenal of the offspring. Exposure to maternal obesity alone also resulted in an increase in adrenal AT1R abundance. There was no effect of maternal dietary restriction or obesity on ACE2 and AT2R or on ERK, calcium/calmodulin-dependent kinase II abundance, and their phosphorylated forms in the lamb adrenal. Thus, weight loss around the time of conception, in both normal-weight and obese ewes, results in changes within the intra-adrenal RAS consistent with increased AT1R activation. These changes within the intra-adrenal RAS system may contribute to the greater adrenal stress response following exposure to signals of adversity in the periconceptional period.

We have postulated that sympathetic neurones may be one source of circulating enkephalins in the fetus. Therefore, we have investigated the effects of increasing gestational age on the content of the enkephalin-containing peptides in the superior cervical ganglia (SCG) of the fetal sheep during late gestation. We have also investigated the role of the pituitary-thyroid axis in the coordinate regulation of enkephalin-containing peptides within the fetal sympathoadrenal system. We found an increase (P0.05) in the content of Free Met-O-Enkephalin between 85 and 105 days (d) (0.46 +/- 0.12 nmoles/SCG) and 116 and 125 d (0.93 +/- 0.12 nmoles/SCG), and the SCG content of Free Met-O-Enkephalin remained elevated throughout the remainder of gestation. The fetal SCG content of Total Met-O-Enkephalin also increased significantly between 85 and 105 d (2.8 +/- 0.5 nmoles/SCG) and 116 and 125 d (7.1 +/- 1.2 nmoles/SCG). There was, therefore, no change in the ratio of Total:Free Met-O-Enkephalin content of the SCG between 85 and 145 d. There were no significant effects of fetal hypophysectomy with or without thyroxine (T4) replacement on the content of Free Met-O-Enkephalin (intact, 1.11 +/- 0.18 nmoles; hypophysectomy (HX) + Sal, 1.41 +/- 0.25 nmoles; HX + T4, 1.08 +/- 0.27 nmoles) or Total Met-O-Enkephalin (intact, 3.03 +/- 0.90 nmoles; HX + Sal, 4.03 +/- 0.22 nmoles; HX + T4, 3.53 +/- 0.88 nmoles) in the fetal SCG. The adrenal contents of Free and Total Met-O-Enkephalin were, however, significantly reduced after fetal hypophysectomy and these effects were not altered by replacement with T4 after fetal hypophysectomy. In summary, we have demonstrated for the first time that enkephalin-containing peptides are present in the SCG of the fetal sheep and that the SCG enkephalin content increases between 85 and 125 days gestation. We have found no evidence that there is a change in the post-translational processing of the enkephalin precursor to Free Met-Enkephalin within the SCG during the second half of gestation. In contrast to the fetal SCG, maintenance of adrenal enkephalin content in late gestation is dependent on the presence of an intact fetal pituitary, but is independent of the circulating concentrations of thyroxine.

1. We have investigated the role of the fetal hypothalamo-pituitary axis in the control of adrenocortical growth and steroidogenesis in the sheep fetus during late gestation. Plasma concentrations of ACTH(1-39) increased between 120-125 and 136-142 days (P < 0.05), but did not change after surgical disconnection of the fetal hypothalamus and pituitary (HPD) at 106-120 days gestation. There was no effect of either gestational age or HPD on the circulating concentrations of the ACTH-containing precursors pro-opiomelanocortin (POMC) and pro-ACTH (the 22 kDa N-terminal portion of POMC). 2. In the fetal sheep adrenal, the relative abundance of the mRNAs of the steroidogenic enzymes CYPIIA1 and CYP21A1 increased between 130-135 and 136-140 days gestation (P < 0.05) and remained high after 141 days, whereas that of CYP17 mRNA increased after 141 days gestation (P < 0.05). The abundance of adrenal 3 beta-HSD mRNA did not change between 130 and 145 days. 3. Hypothalamo-pituitary disconnection significantly reduced the abundance of of CYPIIA1 mRNA, 3 beta-HSD mRNA and CYP17 mRNA by 3.4, 3.1 and 3.7 times, respectively, at 140-142 days gestation (P < 0.05). 4. In the intact group of fetal sheep, adrenal weight increased between 130-135 and 141-145 days (P < 0.05), but there was no change in the abundance of adrenal insulin-like growth factor II (IGF-II) mRNA across this gestational age range. Hypothalamo-pituitary disconnection significantly reduced fetal adrenal weight to 66% that of intact sheep (P < 0.01), but did not alter the abundance of IGF-II mRNA in the fetal adrenal at 140-142 days. 5. Our results suggest that the prepartum changes in adrenal growth and steroidogenesis are under the control of an intact hypothalamo-pituitary axis in late gestation and are dependent on an increase in circulating ACTH(1-39), rather than on ACTH precursors. We have found no evidence, however, for a direct-relationship between fetal adrenal growth or steroidogenesis and adrenal IGF-II mRNA between 130 and 145 days gestation.

We have used the retrograde perfused adrenal preparation to investigate the catecholamine responses of the fetal sheep adrenal to increasing doses of acetylcholine (ACh) and excess potassium. Adrenal glands were collected from fetal sheep between 84 and 99 days gestation (before innervation; n = 7), between 103 and 113 days gestation (during innervation; n = 8) and between 137 and 144 days (after innervation; n = 9). Whilst the basal output of noradrenaline (NA) did not change between 84 and 144 days gestation, there was a significant increase in the adrenal output of adrenaline (Adr) between 84 and 144 days. The NA response to submaximal doses of ACh (10-10(3) microM) was significantly greater (P < 0.05) between 84 and 99 days gestation (mean NA response: 49.6 +/- 11.4 nmol/(30 min)) than after 137 days gestation (mean NA response: 27.3 +/- 8.3 nmol/(30 min)). Similarly, when the NA response to 10-10(3) microM ACh was expressed as a proportion of the maximal NA response to 10(4) microM ACh, the proportional NA response was greater at 84-99 days (48.9 +/- 11.3% of maximal) than at 137-144 days gestation (27.2 +/- 8.3%). The adrenal NA and Adr responses to 10(3) microM ACh between 84 and 137 days were reduced by up to 99% after the addition of hexamethonium. It appears, therefore, that the fetal adrenal is responsive to ACh before the development of innervation of the gland. There is also a decrease in sensitivity of the NA-secreting cells to submaximal doses of ACh as gestation progresses, which is not associated with a decrease in the size of the releasable pool of NA in the fetal adrenal. We speculate, therefore, that innervation of the fetal adrenal may be associated with an increase in the excitation threshold of the NA cells to nicotinic stimulation.

We have investigated the effect of surgical disconnection of the fetal hypothalamus and pituitary (HPD) on generation of the daily rhythm in fetal plasma melatonin and PRL concentrations under long and short photoperiods. Fetal HPD or a sham operation was carried out at around 110 days gestation. Ewes carrying either HPD fetal sheep (n = 10) or intact fetal sheep (n = 12) were exposed to a long light (LL; 16 h of light and 8 h of darkness) or a short light (SL; 8 h of light and 16 h of darkness) regimen for the remainder of gestation. All ewes were subjected to a 24-h blood-sampling experiment (13 samples collected between 0900-0900 h the following day) between 135-140 days gestation, and fetal and maternal plasma melatonin and PRL concentrations were measured using specific RIAs. The hormonal data were analyzed using multifactorial analysis of variance and cosinor analysis. There was an increase in maternal melatonin concentrations during the dark phase in each lighting regimen in ewes carrying HPD or intact fetal sheep. In the SL regimen, there was also a significant increase in fetal melatonin concentrations during the dark phase in the HPD and intact groups. Under LL conditions, however, fetal melatonin concentrations were only consistently increased during the dark phase in the intact, not the HPD, group. The 24-h mean fetal plasma concentrations of PRL were significantly higher (P0.001) in both intact and HPD fetuses in the LL (intact, 111.0 +/- 22.0 pg/ml; HPD, 37.6 +/- 7.3 pg/ml) than in the SL regimen (intact, 37.8 +/- 18.4 pg/ml; HPD, 6.7 +/- 4.3 pg/ml). There was also a significant interaction (P0.001) between the effects of fetal surgical treatment and time of day on fetal PRL concentrations. In the intact group, fetal PRL concentrations were significantly higher (P0.05) at 1300 and 1700 h than between 0300-0700 h in both lighting conditions. Cosinor analysis also identified a significant rhythm in 8 of the 12 fetal PRL profiles in the intact group. In contrast, in the HPD group, there was no significant effect of time of day on fetal PRL in either the LL or SL regimen, and cosinor analysis only identified a significant rhythm in 2 of the 10 fetal PRL profiles in this group. We have, therefore, demonstrated that in the fetal sheep, HPD resulted in abolition of the diurnal melatonin rhythm under LL conditions and in the loss of the diurnal PRL rhythm under LL and SL conditions.(ABSTRACT TRUNCATED AT 400 WORDS)

We compared the effects of the demands of term and preterm infants on the daily rhythms of sleep and wakefulness and salivary melatonin and cortisol concentrations in mothers for up to 5 months after either birth (term group) or arrival of the infant home (preterm group). Although there were relatively small differences between the term and preterm groups in the daily patterns of infant sleep-wake behavior, there were more marked differences in the maternal sleep-wake parameters. During the first 8 weeks after the arrival of the infant home, the mothers of preterm infants had significantly less time asleep and fewer sleep bouts per 24 hours than did the mothers of term infants. The mothers of preterm infants spent a significantly longer proportion of each night awake (30-40%) for the first 8 weeks than did the mothers of the term infants (20-30%). There was also a significant difference between the term and preterm groups in the effect of time of day on maternal salivary melatonin concentrations. In the term group, maternal melatonin concentrations were higher at night (10 p.m.-6 a.m.) than at any other time of day. In contrast, in the preterm group maternal melatonin concentrations between 10 p.m. and 6 a.m. were only higher than those measured between 6 a.m. and 2 p.m. Salivary cortisol concentrations were significantly higher in the mothers of preterm infants than in the mothers of term infants throughout the 10-week study period, but the peak in salivary cortisol concentrations occurred between 2 a.m. and 10 a.m. in both the term and preterm groups.(ABSTRACT TRUNCATED AT 250 WORDS)

We have studied the effects of independently altering the time of darkness and the phase of the daily melatonin rhythm during a 12-h photoperiod on the diurnal rhythms of fetal breathing movements (FBMs), low voltage electrocortical activity, and maternal and fetal plasma concentrations of PRL. Thirteen pregnant ewes were pinealectomized at 99-100 days gestation and held under a normal lighting regime (12-h photoperiod, lights off at 1900 h) until 135 days gestation. All ewes were then exposed to an altered lighting regime (12-h photoperiod, lights off at 1100 h) until 145 days gestation. Six of these ewes were infused with melatonin (12-15 micrograms/h iv) from 1100 h for 12 h each day between 125 and 135 days gestation while in the normal lighting regime, and from 1900 h while in the altered lighting regime. The remaining seven ewes were infused with saline at corresponding times. Infusion of melatonin produced a significant daily rhythm in maternal and fetal melatonin concentrations; concentrations were highest throughout the 12-h infusion period in each lighting regime. In the normal lighting regime (i.e. lights off at 1900 h) the peak incidence of FBM occurred at between 1000 and 1300 h in the ewes infused with saline or melatonin. Under the altered lighting conditions there was no 24-h variation in the incidence of FBMs in the saline-infused group, but a significant daily variation was present in the melatonin-infused group with peak incidence occurring at 1900-2200 h. The daily variation in maternal PRL concentrations was not affected by changes in the time of onset of melatonin infusion but was different in each of the two lighting regimes. In the normal lighting regime, PRL concentrations were highest at 2100 h (56.2 +/- 21.6 ng/ml) whereas in the altered lighting conditions the peak in maternal PRL occurred at 1700 h (90.4 +/- 25.1 ng/ml). The daily variation in fetal PRL concentrations was also not affected by changes in the time of onset of melatonin infusion but was different in the two lighting regimes; in the normal lighting regime, fetal PRL reached a peak (13.0 +/- 3.7 ng/ml) at 0100-0300 h whereas in the altered lighting regime the peak (29.2 +/- 9.8 ng/ml) occurred at 1700-2100 h. We have presented evidence therefore that the daily melatonin rhythm may entrain the daily rhythm in fetal breathing activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Women entering pregnancy with a high body weight and fat mass have babies at increased risk of becoming overweight or obese in childhood and later life. It is not known, whether exposure to a high level of maternal nutrition before pregnancy and exposure to a high transplacental nutrient supply in later pregnancy act through similar mechanisms to program later obesity. Using the pregnant sheep we have shown that maternal overnutrition in late pregnancy results in an upregulation of PPARγ activated genes in fetal visceral fat and a subsequent increase in the mass of subcutaneous fat in the postnatal lamb. Exposure to maternal overnutrition during the periconceptional period alone, however, results in an increase in total body fat mass in female lambs only with a dominant effect on visceral fat depots. Thus the early programming of later obesity may result from 'two hits', the first occurring as a result of maternal overnutrition during the periconceptional period and the second occurring as a result of increased fetal nutrition in late pregnancy. Whilst a short period of dietary restriction during the periconceptional period reverses the impact of periconceptional overnutrition on the programming of obesity, it also results in an increased lamb adrenal weight and cortisol stress response, together with changes in the epigenetic state of the insulin like growth factor 2 (IGF2) gene in the adrenal. Thus, not all of the effects of dietary restriction in overweight or obese mother in the periconceptional period may be beneficial in the longer term.

Women entering pregnancy with a high body weight and fat mass have babies who are at increased risk of becoming overweight or obese in later life. We investigated whether maternal overnutrition in the periconceptional period results in an increased fat mass and expression of adipogenic and lipogenic genes in offspring and whether dietary restriction can reverse these changes. Nonpregnant donor ewes (n = 23) were assigned to one of four groups: control-control fed at 100% maintenance energy requirements (MER) for at least 5 months, control-restricted fed 100% MER for 4 months and 70% MER for 1 month, high-high (HH) fed ad libitum (170–190% MER) for 5 months, or high-restricted (HR) fed ad libitum for 4 months and 70% MER for 1 month. Single embryos were transferred to nonobese recipient ewes, and lamb fat depots were weighed at 4 months. Peroxisome proliferator-activated receptor-γ, glyceraldehyde-3-phosphate dehydrogenase, lipoprotein lipase, leptin, and adiponectin mRNA expression was measured in the lamb fat depots. Total fat mass was higher in female lambs in the HH but not HR group than controls. There was a relationship between donor ewe weight and total fat mass and G3PDH mRNA expression in perirenal fat in female lambs. There was no effect of periconceptional nutritional treatment on peroxisome proliferator-activated receptor-γ, glyceraldehyde-3-phosphate dehydrogenase, lipoprotein lipase, leptin, and adiponectin mRNA expression in any fat depot. Thus, exposure to maternal overnutrition in the periconceptional period alone results in an increased body fat mass in the offspring and that a short period of dietary restriction can reverse this effect.

We have investigated the ACTH and cortisol responses to acute episodes of hypoxemia or hypoglycemia in fetal sheep in which the hypothalamus and pituitary were surgically disconnected at between 112 and 123 days gestation. Before 130 days gestation, basal plasma concentrations of ACTH were significantly greater in the hypothalamo-pituitary disconnected (HPD) fetuses than in the intact fetal sheep (126-130 days; 105.0 +/- 11.4 ng/liter, HPD group; 64.0 +/- 9.5 ng/liter, intact group). After 130 days, however, there was no difference between plasma ACTH concentrations in the HPD (136-140 days; 154.7 +/- 16.7 ng/liter HPD group; 113.6 +/- 19.1 ng/liter, intact group) and intact fetal sheep, and in both groups the mean ACTH concentrations were significantly greater after 136 days gestation than before 130 days. In the HPD group, however, while the plasma ACTH concentrations were elevated there was no prepartum increase in the plasma concentrations of cortisol. A decrease in the fetal arterial blood PO2 by approximately 50% for 30 min between 123 and 132 days, stimulated a significant increase in fetal ACTH and cortisol concentrations in the intact but not in the HPD fetuses. In the intact group, plasma ACTH concentrations were also significantly increased (P less than 0.001) above control values (98.2 +/- 21.2 ng/liter) at 120 min after the start of an iv infusion of insulin (1 IU/60 min) (517.2 +/- 160.5 ng/liter) and were still elevated at 60 min after the end of the infusion period (1248.1 +/- 643.2 ng/liter). In the HPD fetuses, however, there was no significant change in plasma ACTH concentrations during or after the insulin infusion. In both the HPD and intact groups, there was a significant increase in plasma ACTH concentrations above control values (62.5 +/- 8.5 ng/liter intact; 135.0 +/- 30.6 ng/liter, HPD) after intrafetal administration of CRF (+10 min; 117.5 +/- 8.1 ng/liter, intact; 225.3 +/- 33.1 ng/liter, HPD) indicating that the secretory capacity of the pituitary corticotrophs was not reduced by the HPD procedure. Our results demonstrate that an intact hypothalamic-pituitary connection is required to generate a normal prepartum increase in fetal cortisol concentrations and is essential for an appropriate fetal pituitary-adrenal response to intrauterine hypoxemia and hypoglycemia.

Exposure to maternal overnutrition increases the expression of peroxisome proliferator-activated receptor-γ (PPARγ) in adipose tissue before birth, and it has been proposed that the precocial activation of PPARγ target genes may lead to increased fat deposition in postnatal life. In this study, we determined the effect of intrafetal administration of a PPARγ agonist, rosiglitazone, on PPARγ target gene expression in fetal adipose tissue as well indirect actions of rosiglitazone on fetal liver and skeletal muscle. Osmotic pumps containing rosiglitazone (n = 7) or vehicle (15% ethanol, n = 7) were implanted into fetuses at 123–126 d gestation (term = 150 ± 3 d gestation). At 137–141 d gestation, tissues were collected and mRNA expression of PPARγ, lipoprotein lipase (LPL), adiponectin, and glycerol-3-phosphate dehydrogenase (G3PDH) in adipose tissue, PPARα and PPARγ-coactivator 1α (PGC1α) in liver and muscle and phosphoenolpyruvate carboxykinase (PEPCK) in liver determined by quantitative real-time RT-PCR. Plasma insulin concentrations were lower in rosiglitazone-treated fetuses (P < 0.02). Rosiglitazone treatment resulted in increased expression of LPL and adiponectin mRNA (P < 0.01) in fetal adipose tissue. The expression of PPARα mRNA in liver (P < 0.05) and PGC1α mRNA (P < 0.02) in skeletal muscle were also increased by rosiglitazone treatment. Rosiglitazone treatment increased expression of PPARγ target genes within fetal adipose tissue and also had direct or indirect actions on the fetal liver and muscle. The effects of activating PPARγ in fetal adipose tissue mimic those induced by prenatal overnutrition, and it is therefore possible that activation of PPARγ may be the initiating mechanism in the pathway from prenatal overnutrition to postnatal obesity.

It is unclear whether the maturation of corticotrophs from the fetal to the adult type in the fetal sheep pituitary in late gestation is associated with changes in the sensitivity of the fetal pituitary to corticotrophic secretagogues and in the form of ACTH-containing peptides (IR-ACTH) secreted into the circulation. The maturation of the pituitary corticotroph population is known to be accelerated by intrafetal cortisol infusion and delayed by bilateral fetal adrenalectomy. We have therefore investigated the mol wt profile of IR-ACTH present in fetal sheep plasma from 110 days gestation until term (147 +/- 3 days) and determined whether intrafetal cortisol infusion between 105-117 days (2.5 mg cortisol/day), or bilateral fetal adrenalectomy can alter the mol wt profile of IR-ACTH in fetal sheep plasma. We have also investigated whether prior exposure to cortisol alters the subsequent responsiveness of the fetal pituitary to a long term infusion of ovine (o) CRF (10 micrograms oCRF/day). In the control group, the proportion of IR-ACTH which eluted in the low-mol wt (LMW) range (i.e. less than 12K) was significantly higher between 121-125 days (43.9 +/- 4.2%) than between 126-139 days (26.8 +/- 9.3%) but not different to that after 140 days gestation (29.9 +/- 5.5%). Between 110-117 days, cortisol infusion had no effect on the proportion of IR-ACTH in the LMW range (43.9 +/- 5.7%, saline infused; 44.1 +/- 2.4%, cortisol infused). Between 121-125 days, the proportion of IR-ACTH in the LMW range in the CRF-infused groups (with or without prior exposure to cortisol) was significantly lower (27.4 +/- 2.1%) than in the saline-infused control group. In contrast, after fetal adrenalectomy, the proportion of IR-ACTH in the LMW range between 126-139 days was significantly higher (48.0 +/- 6.7%) than in intact control animals (23.8 +/- 3.5%). We conclude that the change in the mol wt profile of IR-ACTH in fetal plasma after 125 days may be a consequence of changes in the morphological and/or functional characteristics of the corticotrophic cells in the fetal pituitary. Infusion of oCRF appears to accelerate the normal maturation of the fetal pituitary-adrenal relationship, and oCRF acting either directly or via secretion of cortisol may play a role in the posttranslational processing of POMC in the fetal sheep pituitary after 125 days gestation.

The effects of two different feeding regimes on the 24 h profiles of maternal and fetal plasma cortisol and adrenocorticotrophic hormone (ACTH) concentrations were studied in eight pregnant ewes between 123 and 144 days of gestation. Once daily-fed ewes (n = 4) received 1 kg of lucerne-chaff at 11.00 h, and multi-fed ewes (n = 4) received 100-200 g of lucerne-chaff at 09.00, 11.00 and 13.00 h and then 150 g until 09.00 h the following day. There were significant differences between the two feeding groups in the 24 h profile of maternal plasma osmolality; once daily feeding at 11.00 h was associated with a peak in maternal plasma osmolality at 15.00 h whereas maternal plasma osmolality reached plateau levels at around 17.00 h in the multi-fed group. There were also differences between the two feeding groups in the 24 h profiles of maternal and fetal plasma glucose. Maternal and fetal plasma glucose reached peak concentrations at 19.00 h in the once daily-fed ewes in contrast to the multi-fed group, where a plateau in maternal and fetal plasma glucose was reached between 19.00 h and 09.00 h the following day. A significant diurnal variation in the plasma concentrations of cortisol was present in the once daily-fed ewes from 123 days gestation and in their fetuses after, but not before, 135 days gestation. Plasma cortisol peaked at 11.00 h in the ewes and at 13.00 h in the fetuses of this group. In the once daily-fed group there was also a significant diurnal variation in maternal and fetal plasma ACTH; plasma ACTH concentrations were highest at 11.00 h in the ewes aged between 123 and 144 days and in fetuses after 135 days gestation. In the multi-fed group, whilst ACTH was highest at 09.00 h in the ewes and at 13.00 h in the fetuses, there was no significant diurnal variation in the plasma concentrations of cortisol in the ewes or fetuses of this group at any stage between 123 and 144 days gestation.

Epidemiological studies have demonstrated that low birth weight is associated with an increased incidence of visceral obesity and metabolic disorders in later life. In the present study, we have determined the impact of birth weight and gender on gene expression in visceral adipose tissue (VAT) in the young adult sheep. Lambs ( n =19, birth weight range 2.6–7.55kg) were born at term and growth monitored for 22.4±0.2 weeks, when body composition was determined by Dual X-ray Absorptiometry (DXA) and samples of VAT and subcutaneous (SCAT) adipose tissue collected. Plasma samples were collected at post-mortem for the determination of free fatty acids (FFA), glucose and insulin concentrations. Peroxisome-Proliferator Activated Receptor-γ (PPARγ), glycerol-3-phosphate dehydrogenase (G3PDH), lipoprotein lipase (LPL), adiponectin and leptin mRNA expression was determined by qRT-PCR. Fractional growth rate in postnatal weeks 1–3 was inversely related to birth weight in both males and females ( R 2 =0.22, P n =19). PPARγ mRNA expression in VAT, but not SCAT, was inversely related to birth weight ( R 2 =0.60, P n =18). In males, but not females, PPARγ mRNA in VAT was directly related to G3PDH mRNA expression ( R 2 =0.69, P n =9). Plasma FFA concentrations were inversely related to birth weight in both males and females ( R 2 =0.22, P n =19). These findings demonstrate that low birth weight is associated with an increased expression of a key adipogenic factor in visceral adipose tissue in young adulthood. In males, this is associated with an increased expression of lipogenic genes, and this may contribute to the increased propensity for visceral obesity in low birth weight males compared to females.

Our aim was to determine fetal and maternal endocrine and fluid-balance responses to prolonged loss of amniotic and allantoic fluids in sheep. In seven sheep, amniotic and allantoic fluids were drained [379.1 +/- 20.1 (SE) ml/day] from 107 to 135.3 +/- 0.6 days of gestation (term: 145 days). The results from these sheep were compared with those from seven control sheep. Maternal water intake, urine production, and urine osmolality were not altered by fluid drainage, nor were fetal and maternal arterial blood gases, pH, or plasma osmolalities. Fluid drainage increased amniotic, but not allantoic, fluid osmolality. Maternal plasma cortisol concentration increased with fluid drainage, but maternal plasma concentrations of prolactin and arginine vasopressin were unchanged. Fluid drainage increased prolactin concentrations in fetal plasma and amniotic fluid, but fetal plasma concentrations of cortisol (hydrocortisone), arginine vasopressin, norepinephrine, and epinephrine were unchanged. Our results show that the fetus is capable of maintaining its plasma osmolality despite prolonged loss of fluid from its amniotic and allantoic sacs and that this is associated with alterations in the production rate and the composition of amniotic fluid.

We have monitored the 24-h profiles of plasma melatonin concentrations between birth and 10 weeks of age, in lambs which were delivered to, and suckled, either pineal-intact (control group) or pinealectomized (pinealectomized group) ewes. Between 0 and 2 weeks of age, plasma concentrations of melatonin in lambs suckling either intact or pinealectomized ewes were highest at 01.00 h. At this age, however, there was no significant difference in the mean plasma concentrations of melatonin between the entire dark and light phases in lambs in either the control group (dark, 39·7 ± 6·0 (s.e.m.) pmol/l; light, 39·5 ± 8·1 pmol/l) or the pinealectomized group (dark, 79·8 ± 43·3 pmol/l; light, 60·9 ±8·7 pmol/l). Between 3 and 4 weeks of age, however, a diurnal rhythm in plasma melatonin concentrations was clearly present in the lambs in both the control and pinealectomized groups (control group: dark, 164·1 ± 5·6 pmol/l; light 26·2 ± 2·5 pmol/l; pinealectomized group: dark, 52·7± 8·0 pmol/l; light, 19·1 ± 5·3 pmol/l; PPP In conclusion, we have demonstrated that a clear diurnal plasma rhythm in melatonin concentrations does not emerge until 3–4 weeks of age in lambs suckling either pinealectomized or intact ewes. We have also measured lower melatonin concentrations during the dark phase in lambs suckling pinealectomized ewes when compared with lambs suckling pineal-intact ewes between 3 and 10 weeks of age. Journal of Endocrinology (1990) 125, 97–102

We have investigated the effect of pinealectomy of the pregnant ewe on the 24-h pattern of fetal breathing activity during late gestation. Fetal breathing movements were recorded during 24-h periods on 18 occasions in 5 pinealectomized ewes and on 24 occasions in 6 pineal-intact ewes between 120 and 145 days gestation. All ewes were fed once daily between 1000 and 1300 h and were kept under a light-dark cycle 12:12 h. There was no significant difference in the mean hourly incidence of fetal breathing movements between the pineal-intact (27.2 +/- 0.5 min/h) and pinealectomized (25.5 +/- 0.6 min/h) groups. However, there was a significant difference in the 24-h profiles of fetal breathing movements in the two groups. The peak incidence of fetal breathing occurred between 1900 and 2000 h in the pineal-intact ewes and between 1200 and 1300 h in the pinealectomized ewes. We conclude that maternal pinealectomy alters the daily pattern of a fetal behavioral pattern and that maternal melatonin may therefore provide the fetus with information about time of day.

Prenatal and early postnatal life experiences, reflected by size at birth and postnatal catch-up growth, contribute to the risk of developing the metabolic syndrome in adulthood, but their relative importance is unclear. Therefore, we determined the effects of restricted placental and fetal growth on components of the metabolic syndrome in young adult sheep and the relationships of the latter to size at birth and early postnatal growth. Fasting plasma metabolites, glucose tolerance (by intravenous glucose tolerance test, IVGTT), insulin secretion and sensitivity, and resting blood pressure were measured in 22 control and 20 placentally restricted (PR) 1-yr-old sheep. In male sheep, PR increased the initial rise in glucose during an IVGTT and reduced diastolic blood pressure, and small size at birth independently predicted reduced adult size, glucose tolerance, and fasting plasma insulin and insulin disposition of glucose metabolism but increased insulin disposition of circulating FFAs. Also in males, high fractional growth rates in early postnatal life independently predicted impaired early glucose clearance during an IVGTT. In female animals, PR increased insulin sensitivity of glucose metabolism and reduced fasting plasma FFAs, and thinness at birth predicted increased adult size, fasting blood glucose, and pulse pressure. In conclusion, PR and small size at birth are associated with more components of the metabolic syndrome in adult male than in adult female sheep, with few independent effects of early postnatal growth. These sex differences in the onset and extent of adverse metabolic consequences after prenatal restraint in the sheep are consistent with observations in humans.

The efficiency of cloning by somatic cell nuclear transfer (SCNT) is poor in livestock with ~5% of transferred cloned embryos developing to term. SCNT is associated with gross placental structural abnormalities. We aimed to identify defects in placental histology and gene expression in failing ovine cloned pregnancies to better understand why so many clones generated by SCNT diein utero. Placentomes from SCNT pregnancies (n= 9) and age matched, naturally mated controls (n= 20) were collected at two gestational age ranges (105–134 days and 135–154 days; term = 147 days). There was no effect of cloning on total placental weight. However, cloning reduced the number of placentomes at both gestational ages (105–134 days: control 55.0 ± 4.2, clone 44.7 ± 8.0 and 135–154 days: control 72.2 ± 5.1, clone 36.6 ± 5.1;P< 0.001) and increased the mean individual placentome weight (105–134 days: control 10.6 ± 1.3 g, clone 18.6 ± 2.8 g and 135–154 days: control 6.6 ± 0.6 g, clone 7.0 ± 2.0 g;P< 0.02). Placentomes from cloned pregnancies had a significant volume of shed trophoblast and fetal villous hemorrhage, absent in controls, at both gestational age ranges (P< 0.001) that was shown to be apoptotic by activated caspase-3 immunoreactivity. Consequently, the volume of intact trophoblast was reduced and the arithmetic mean barrier thickness of trophoblast through which exchange occurs was altered (P< 0.001) at both gestational age ranges in clones. In addition, cloning reduced placental expression of key genes in placental differentiation and function. Thus, cloning by SCNT results in both gross and microscopic placental abnormalities. We speculate that trophoblast apoptosis, shedding, and hemorrhage may be causal in fetal death in ovine clones.