An ovine model for investigation of the microenvironment of the male mammary gland.

The specific biology of the male breast remains relatively unexplored in spite of the increasing global prevalence of male breast cancer. Delineation of the microenvironment of the male breast is restricted by the low availability of human samples and a lack of characterisation of appropriate animal models. Unlike the mouse, the male ovine gland persists postnatally. We suggest that the male ovine mammary gland constitutes a promising adjunctive model for the male breast. In this study, we evaluate the male ovine mammary gland microenvironment, comparing intact and neutered males. Assessment of the glandular histo-anatomy highlights the resemblance of the male gland to that of neonatal female sheep and confirms the presence of rudimentary terminal duct lobular units. Irrespective of neutered status, cell proliferation in epithelial and stromal compartments is similarly low in males, and cell proliferation in epithelial cells and in the intralobular stroma is significantly lower than in pubertal female sheep. Between 42% and 72% of the luminal mammary epithelial cells in the male gland express the androgen receptor and expression is significantly reduced by neutering. Luminal epithelial cells within the intact and neutered male gland also express oestrogen receptor alpha, but minimal progesterone receptor expression is observed. The distribution of leukocytes within the ducts and stroma is similar to the mammary gland of female sheep and females of other species. Both macrophages and T lymphocytes are intercalated in the epithelial bilayer and are more abundant in the intralobular stroma than the interlobular stroma, suggesting that they may have a protective immunological function within the vestigial glandular tissue of the male sheep. Mast cells are also observed within the stroma. These cells cluster near the glandular tissue and are frequently located adjacent to blood vessels. The abundance of mast cells is significantly higher in intact males compared to neutered males, suggesting that hormone signalling may impact mast cell recruitment. In this study, we demonstrate the utility of the male ovine mammary gland as a model for furthering our knowledge of postnatal male mammary biology.

Obesogenic diet in pregnancy disrupts placental iron handling and ferroptosis and stress signalling in association with fetal growth alterations.

Obesity and gestational diabetes (GDM) impact fetal growth during pregnancy. Iron is an essential micronutrient needed for energy-intense feto-placental development, but if mis-handled can lead to oxidative stress and ferroptosis (iron-dependent cell death). In a mouse model showing maternal obesity and glucose intolerance, we investigated the association of materno-fetal iron handling and placental ferroptosis, oxidative damage and stress signalling activation with fetal growth. Female mice were fed a standard chow or high fat, high sugar (HFHS) diet during pregnancy and outcomes were measured at day (d)16 or d19 of pregnancy. In HFHS-fed mice, maternal hepcidin was reduced and iron status maintained (tissue iron levels) at both d16 and d19. However, fetal weight, placental iron transfer capacity, iron deposition, TFR1 expression and ERK2-mediated signalling were reduced and oxidative damage-related lipofuscin accumulation in the placenta was increased in HFHS-fed mice. At d19, whilst TFR1 remained decreased, fetal weight was normal and placental weight, iron content and iron transporter genes (Dmt1, Zip14, and Fpn1) were reduced in HFHS-fed mice. Furthermore, there was stress kinase activation (increased phosphorylated p38MAPK, total ERK and JNK) in the placenta from HFHS-fed mice at d19. In summary, a maternal HFHS diet during pregnancy impacts fetal growth trajectory in association with changes in placental iron handling, ferroptosis and stress signalling. Downregulation of placental iron transporters in HFHS mice may protect the fetus from excessive oxidative iron. These findings suggest a role for alterations in placental iron homeostasis in determining perinatal outcomes of pregnancies associated with GDM and/or maternal obesity.

Sex-Specific Effects of a Maternal Obesogenic Diet High in Fat and Sugar on Offspring Adiposity, Growth, and Behavior.

With rising rates of human obesity, this study aimed to determine the relationship between maternal diet-induced obesity, offspring morphometrics, and behavior in mice. Pregnant and lactating female mice fed a diet high in fat and sugar (HFHS) commonly consumed by human populations showed decreased food, calorie, and protein intake but increased adiposity at the expense of lean mass. The pre-weaning body weight of the HFHS offspring was reduced for the first postnatal week but not thereafter, with HFHS female offspring having higher body weights by weaning due to continuing higher fractional growth rates. Post-weaning, there were minor differences in offspring food and protein intake. Maternal diet, however, affected fractional growth rate and total body fat content of male but not female HFHS offspring. The maternal diet did not affect the offspring's locomotor activity or social behavior in either sex. Both the male and female HFHS offspring displayed reduced anxiety-related behaviors, with sex differences in particular aspects of the elevated plus maze task. In the novel object recognition task, performance was impaired in the male but not female HFHS offspring. Collectively, the findings demonstrate that maternal obesity alters the growth, adiposity, and behavior of male and female offspring, with sex-specific differences.

Age and an obesogenic diet affect mouse behaviour in a sex-dependent manner.

Obesity is rising globally and is associated with neurodevelopmental and psychiatric disorders among children, adolescents and young adults. Whether obesity is the cause or the consequence of these disorders remains unclear. To examine the behavioural effects of obesity systematically, locomotion, anxiety and social behaviour were assessed in male and female C57Bl/6J mice using the open field, elevated plus maze and social preference task. First, the effects of age and sex were examined in control mice, before investigating post-weaning consumption of a high fat-high sugar diet commonly consumed in human populations with high rates of obesity. In the open field and elevated plus maze, locomotor activity and anxiety-related behaviours reduced with aging in both sexes, but with different sex-specific profiles. The high fat-high sugar diet reduced food and calorie intake and increased body mass and fat deposition in both sexes. In the open field, both male and female mice on the obesogenic diet showed reduced locomotion; whereas, in the elevated plus maze, only females fed with the obesogenic diet displayed reduced anxiety-related behaviours. Both male and female mice on the obesogenic diet had a significantly higher social preference index than the control group. In conclusion, the findings demonstrate that the behavioural effects of age and diet-induced obesity all depend on the sex of the mouse. This emphasises the importance of considering the age of the animal and including both sexes when assessing behavioural phenotypes arising from dietary manipulations.

Hypothyroidism impairs development of the gastrointestinal tract in the ovine fetus.

Growth and maturation of the fetal gastrointestinal tract near term prepares the offspring for the onset of enteral nutrition at birth. Structural and functional changes are regulated by the prepartum rise in cortisol in the fetal circulation, although the role of the coincident rise in plasma tri-iodothyronine (T3) is unknown. This study examined the effect of hypothyroidism on the structural development of the gastrointestinal tract and the activity of brush-border digestive enzymes in the ovine fetus near term. In intact fetuses studied between 100 and 144 days of gestation (dGA; term ∼145 days), plasma concentrations of T3, cortisol and gastrin; the mucosal thickness in the abomasum, duodenum, jejunum and ileum; and intestinal villus height and crypt depth increased with gestational age. Removal of the fetal thyroid gland at 105-110 dGA suppressed plasma thyroxine (T4) and T3 concentrations to the limit of assay detection in fetuses studied at 130 and 144 dGA, and decreased plasma cortisol and gastrin near term, compared to age-matched intact fetuses. Hypothyroidism was associated with reductions in the relative weights of the stomach compartments and small intestines, the outer perimeter of the intestines, the thickness of the gastric and intestinal mucosa, villus height and width, and crypt depth. The thickness of the mucosal epithelial cell layer and muscularis propria in the small intestines were not affected by gestational age or treatment. Activities of the brush border enzymes varied with gestational age in a manner that depended on the enzyme and region of the small intestines studied. In the ileum, maltase and dipeptidyl peptidase IV (DPPIV) activities were lower, and aminopeptidase N (ApN) were higher, in the hypothyroid compared to intact fetuses near term. These findings highlight the importance of thyroid hormones in the structural and functional development of the gastrointestinal tract near term, and indicate how hypothyroidism in utero may impair the transition to enteral nutrition and increase the risk of gastrointestinal disorders in the neonate.

Developmental programming of mitochondrial substrate metabolism in skeletal muscle of adult sheep by cortisol exposure before birth.

Prenatal glucocorticoid overexposure causes adult metabolic dysfunction in several species but its effects on adult mitochondrial function remain largely unknown. Using respirometry, this study examined mitochondrial substrate metabolism of fetal and adult ovine biceps femoris (BF) and semitendinosus (ST) muscles after cortisol infusion before birth. Physiological increases in fetal cortisol concentrations pre-term induced muscle- and substrate-specific changes in mitochondrial oxidative phosphorylation capacity in adulthood. These changes were accompanied by muscle-specific alterations in protein content, fibre composition and abundance of the mitochondrial electron transfer system (ETS) complexes. In adult ST, respiration using palmitoyl-carnitine and malate was increased after fetal cortisol treatment but not with other substrate combinations. There were also significant increases in protein content and reductions in the abundance of all four ETS complexes, but not ATP synthase, in the ST of adults receiving cortisol prenatally. In adult BF, intrauterine cortisol treatment had no effect on protein content, respiratory rates, ETS complex abundances or ATP synthase. Activity of citrate synthase, a marker of mitochondrial content, was unaffected by intrauterine treatment in both adult muscles. In the ST but not BF, respiratory rates using all substrate combinations were significantly lower in the adults than fetuses, predominantly in the saline-infused controls. The ontogenic and cortisol-induced changes in mitochondrial function were, therefore, more pronounced in the ST than BF muscle. Collectively, the results show that fetal cortisol overexposure programmes mitochondrial substrate metabolism in specific adult muscles with potential consequences for adult metabolism and energetics.

Ablation of PI3K-p110alpha Impairs Maternal Metabolic Adaptations to Pregnancy.

Pregnancy requires adaptations in maternal metabolism to support fetal growth. The phosphoinositol-3-kinase (PI3K) signalling pathway controls multiple biological processes and defects in this pathway are linked to metabolic disorders including insulin resistance and glucose intolerance in non-pregnant animals. However, relatively little is known about the contribution of PI3K signalling to the maternal metabolic adaptations during pregnancy. Using mice with partial inactivation of the PI3K isoform, p110α (due to a heterozygous dominant negative mutation; Pik3ca-D933A), the effects of impaired PI3K-p110α signalling on glucose and insulin handling were examined in the pregnant and non-pregnant states and related to the morphological, molecular, and mitochondrial changes in key metabolic organs. The results show that non-pregnant mice lacking PI3K-p110α are glucose intolerant but exhibit compensatory increases in pancreatic glucose-stimulated insulin release and adipose tissue mitochondrial respiratory capacity and fatty acid oxidation. However, in pregnancy, mutant mice failed to show the normal increment in glucose intolerance and pancreatic ß-cell mass observed in wild-type pregnant dams and exhibited further enhanced adipose tissue mitochondrial respiratory capacity. These maladaptations in pregnant mutant mice were associated with fetal growth restriction. Hence, PI3K-p110α is a key regulator of metabolic adaptations that support fetal growth during normal pregnancy.

Cortisol Regulates Cerebral Mitochondrial Oxidative Phosphorylation and Morphology of the Brain in a Region-Specific Manner in the Ovine Fetus.

In adults, glucocorticoids are stress hormones that act, partly, through actions on mitochondrial oxidative phosphorylation (OXPHOS) to increase energy availability. Before birth, glucocorticoids are primarily maturational signals that prepare the fetus for new postnatal challenges. However, the role of the normal prepartum glucocorticoid rise in preparing mitochondria for the increased postnatal energy demands remains largely unknown. This study examined the effect of physiological increases in the fetal cortisol concentration on cerebral mitochondrial OXPHOS capacity near term (~130 days gestation, term ~145 days gestation). Fetal sheep were infused with saline or cortisol for 5 days at ~0.8 of gestation before the mitochondrial content, respiratory rates, abundance of the electron transfer system proteins and OXPHOS efficiency were measured in their cortex and cerebellum. Cerebral morphology was assessed by immunohistochemistry and stereology. Cortisol treatment increased the mitochondrial content, while decreasing Complex I-linked respiration in the cerebellum. There was no effect on the cortical mitochondrial OXPHOS capacity. Cortisol infusion had regional effects on cerebral morphology, with increased myelination in the cerebrum. The findings demonstrate the importance of cortisol in regulating the cerebral mitochondrial OXPHOS capacity prenatally and have implications for infants born preterm or after glucocorticoid overexposure due to pregnancy complications or clinical treatment.

Metabolic Consequences of Glucocorticoid Exposure before Birth.

Glucocorticoids have an important role in development of the metabolic phenotype in utero. They act as environmental and maturational signals in adapting feto-placental metabolism to maximize the chances of survival both before and at birth. They influence placental nutrient handling and fetal metabolic processes to support fetal growth, fuel storage and energy production with respect to nutrient availability. More specifically, they regulate the transport, utilization and production of a range of nutrients by the feto-placental tissues that enables greater metabolic flexibility in utero while minimizing any further drain on maternal resources during periods of stress. Near term, the natural rise in fetal glucocorticoid concentrations also stimulates key metabolic adaptations that prepare tissues for the new energy demanding functions after birth. Glucocorticoids, therefore, have a central role in the metabolic communication between the mother, placenta and fetus that optimizes offspring metabolic phenotype for survival to reproductive age. This review discusses the effects of maternal and fetal glucocorticoids on the supply and utilization of nutrients by the feto-placental tissues with particular emphasis on studies using quantitative methods to assess metabolism in rodents and sheep in vivo during late pregnancy. It considers the routes of glucocorticoid overexposure in utero, including experimental administration of synthetic glucocorticoids, and the mechanisms by which these hormones control feto-placental metabolism at the molecular, cellular and systems levels. It also briefly examines the consequences of intrauterine glucocorticoid overexposure for postnatal metabolic health and the generational inheritance of metabolic phenotype.

Diet-induced maternal obesity impacts feto-placental growth and induces sex-specific alterations in placental morphology, mitochondrial bioenergetics, dynamics, lipid metabolism and oxidative stress in mice.

AIM: The current study investigated the impact of maternal obesity on placental phenotype in relation to fetal growth and sex. METHODS: Female C57BL6/J mice were fed either a diet high in fat and sugar or a standard chow diet, for 6 weeks prior to, and during, pregnancy. At day 19 of gestation, placental morphology and mitochondrial respiration and dynamics were assessed using high-resolution respirometry, stereology, and molecular analyses. RESULTS: Diet-induced maternal obesity increased the rate of small for gestational age fetuses in both sexes, and increased blood glucose concentrations in offspring. Placental weight, surface area, and maternal blood spaces were decreased in both sexes, with reductions in placental trophoblast volume, oxygen diffusing capacity, and an increased barrier to transfer in males only. Despite these morphological changes, placental mitochondrial respiration was unaffected by maternal obesity, although the influence of fetal sex on placental respiratory capacity varied between dietary groups. Moreover, in males, but not females, maternal obesity increased mitochondrial complexes (II and ATP synthase) and fission protein DRP1 abundance. It also reduced phosphorylated AMPK and capacity for lipid synthesis, while increasing indices of oxidative stress, specifically in males. In females only, placental mitochondrial biogenesis and capacity for lipid synthesis, were both enhanced. The abundance of uncoupling protein-2 was decreased by maternal obesity in both fetal sexes. CONCLUSION: Maternal obesity exerts sex-dependent changes in placental phenotype in association with alterations in fetal growth and substrate supply. These findings may inform the design of personalized lifestyle interventions or therapies for obese pregnant women.

Endocrine regulation of fetal metabolism towards term.

Hormones have an important role in regulating fetal metabolism in relation to the prevailing nutritional conditions both in late gestation and during the prepartum period as the fetus prepares for birth. In particular, the pancreatic, thyroid and adrenal hormones all affect fetal uptake and utilization of nutrients for oxidative metabolism, tissue accretion and fuel storage. These hormones also influence the fetal metabolic preparations for the nutritional transition from intra- to extra-uterine life. This review discusses the role of insulin, glucagon, thyroxine, tri-iodothyronine, cortisol and the catecholamines in these processes during normal intrauterine conditions and in response to maternal undernutrition with particular emphasis on the sheep fetus. It also considers the metabolic interactions between these hormones and their role in the maturation of key tissues, such as the liver, skeletal muscle and adipose tissue, in readiness for their new metabolic functions after birth. Endocrine regulation of fetal metabolism is shown to be multifactorial and dynamic with a central role in optimizing metabolic fitness for survival both in utero and at birth.

The imprinted Igf2-Igf2r axis is critical for matching placental microvasculature expansion to fetal growth.

In all eutherian mammals, growth of the fetus is dependent upon a functional placenta, but whether and how the latter adapts to putative fetal signals is currently unknown. Here, we demonstrate, through fetal, endothelial, hematopoietic, and trophoblast-specific genetic manipulations in the mouse, that endothelial and fetus-derived IGF2 is required for the continuous expansion of the feto-placental microvasculature in late pregnancy. The angiocrine effects of IGF2 on placental microvasculature expansion are mediated, in part, through IGF2R and angiopoietin-Tie2/TEK signaling. Additionally, IGF2 exerts IGF2R-ERK1/2-dependent pro-proliferative and angiogenic effects on primary feto-placental endothelial cells ex vivo. Endothelial and fetus-derived IGF2 also plays an important role in trophoblast morphogenesis, acting through Gcm1 and Synb. Thus, our study reveals a direct role for the imprinted Igf2-Igf2r axis on matching placental development to fetal growth and establishes the principle that hormone-like signals from the fetus play important roles in controlling placental microvasculature and trophoblast morphogenesis.

Pancreas deficiency modifies bone development in the ovine fetus near term.

Hormones have an important role in the regulation of fetal growth and development, especially in response to nutrient availability in utero. Using micro-CT and an electromagnetic three-point bend test, this study examined the effect of pancreas removal at 0.8 fraction of gestation on the developing bone structure and mechanical strength in fetal sheep. When fetuses were studied at 10 and 25 days after surgery, pancreatectomy caused hypoinsulinaemia, hyperglycaemia and growth retardation which was associated with low plasma concentrations of leptin and a marker of osteoclast activity and collagen degradation. In pancreatectomized fetuses compared to control fetuses, limb lengths were shorter, and trabecular (Tb) bone in the metatarsi showed greater bone volume fraction, Tb thickness, degree of anisotropy and porosity, and lower fractional bone surface area and Tb spacing. Mechanical strength testing showed that pancreas deficiency was associated with increased stiffness and a greater maximal weight load at fracture in a subset of fetuses studied near term. Overall, pancreas deficiency in utero slowed the growth of the fetal skeleton and adapted the developing bone to generate a more compact and connected structure. Maintenance of bone strength in growth-retarded limbs is especially important in a precocial species in preparation for skeletal loading and locomotion at birth.

Developing ovine mammary terminal duct lobular units have a dynamic mucosal and stromal immune microenvironment.

The human breast and ovine mammary gland undergo striking levels of postnatal development, leading to formation of terminal duct lobular units (TDLUs). Here we interrogate aspects of sheep TDLU growth as a model of breast development and to increase understanding of ovine mammogenesis. The distributions of epithelial nuclear Ki67 positivity differ significantly between younger and older lambs. Ki67 expression is polarised to the leading edge of the developing TDLUs. Intraepithelial ductal macrophages exhibit periodicity and considerably increased density in lambs approaching puberty. Stromal macrophages are more abundant centrally than peripherally. Intraepithelial T lymphocytes are more numerous in older lambs. Stromal hotspots of Ki67 expression colocalize with immune cell aggregates that exhibit distinct organisation consistent with tertiary lymphoid structures. The lamb mammary gland thus exhibits a dynamic mucosal and stromal immune microenvironment and constitutes a valuable model system that provides new insights into postnatal breast development.

Deletion of the Imprinted Phlda2 Gene Increases Placental Passive Permeability in the Mouse.

Genomic imprinting, an epigenetic phenomenon that causes the expression of a small set of genes in a parent-of-origin-specific manner, is thought to have co-evolved with placentation. Many imprinted genes are expressed in the placenta, where they play diverse roles related to development and nutrient supply function. However, only a small number of imprinted genes have been functionally tested for a role in nutrient transfer capacity in relation to the structural characteristics of the exchange labyrinthine zone. Here, we examine the transfer capacity in a mouse model deficient for the maternally expressed Phlda2 gene, which results in placental overgrowth and a transient reduction in fetal growth. Using stereology, we show that the morphology of the labyrinthine zone in Phlda2-/+ mutants is normal at E16 and E19. In vivo placental transfer of radiolabeled solutes 14C-methyl-D-glucose and 14C-MeAIB remains unaffected at both gestational time points. However, placental passive permeability, as measured using two inert hydrophilic solutes (14C-mannitol; 14C-inulin), is significantly higher in mutants. Importantly, this increase in passive permeability is associated with fetal catch-up growth. Our findings uncover a key role played by the imprinted Phlda2 gene in modifying placental passive permeability that may be important for determining fetal growth.

Development of cerebral mitochondrial respiratory function is impaired by thyroid hormone deficiency before birth in a region-specific manner.

Thyroid hormones regulate adult metabolism partly through actions on mitochondrial oxidative phosphorylation (OXPHOS). They also affect neurological development of the brain, but their role in cerebral OXPHOS before birth remains largely unknown, despite the increase in cerebral energy demand during the neonatal period. Thus, this study examined prepartum development of cerebral OXPHOS in hypothyroid fetal sheep. Using respirometry, Complex I (CI), Complex II (CII), and combined CI&CII OXPHOS capacity were measured in the fetal cerebellum and cortex at 128 and 142 days of gestational age (dGA) after surgical thyroidectomy or sham operation at 105 dGA (term ~145 dGA). Mitochondrial electron transfer system (ETS) complexes, mRNA transcripts related to mitochondrial biogenesis and ATP production, and mitochondrial density were quantified using molecular techniques. Cerebral morphology was assessed by immunohistochemistry and stereology. In the cortex, hypothyroidism reduced CI-linked respiration and CI abundance at 128 dGA and 142 dGA, respectively, and caused upregulation of PGC1α (regulator of mitochondrial biogenesis) and thyroid hormone receptor ß at 128 dGA and 142 dGA, respectively. In contrast, in the cerebellum, hypothyroidism reduced CI&II- and CII-linked respiration at 128 dGA, with no significant effect on the ETS complexes. In addition, cerebellar glucocorticoid hormone receptor and adenine nucleotide translocase (ANT1) were downregulated at 128 dGA and 142 dGA, respectively. These alterations in mitochondrial function were accompanied by reduced myelination. The findings demonstrate the importance of thyroid hormones in the prepartum maturation of cerebral mitochondria and have implications for the etiology and treatment of the neurodevelopmental abnormalities associated with human prematurity and congenital hypothyroidism.

Thyroid Hormone Deficiency Suppresses Fetal Pituitary-Adrenal Function Near Term: Implications for the Control of Fetal Maturation and Parturition.

Background: The fetal hypothalamic-pituitary-adrenal (HPA) axis plays a key role in the control of parturition and maturation of organ systems in preparation for birth. In hypothyroid fetuses, gestational length may be prolonged and maturational processes delayed. The extent to which the effects of thyroid hormone deficiency in utero on the timing of fetal maturation and parturition are mediated by changes to the structure and function of the fetal HPA axis is unknown. Methods: In twin sheep pregnancies where one fetus was thyroidectomized and the other sham-operated, this study investigated the effect of hypothyroidism on circulating concentrations of adrenocorticotrophic hormone (ACTH) and cortisol, and the structure and secretory capacity of the anterior pituitary and adrenal glands. The relative population of pituitary corticotrophs and the masses of the adrenal zones were assessed by immunohistochemical and stereological techniques. Adrenal mRNA abundances of key steroidogenic enzymes and growth factors were examined by quantitative polymerase chain reaction. Results: Hypothyroidism in utero reduced plasma concentrations of ACTH and cortisol. In thyroid-deficient fetuses, the mass of corticotrophs in the anterior pituitary gland was unexpectedly increased, while the mass of the zona fasciculata and its proportion of the adrenal gland were decreased. These structural changes were associated with lower adrenocortical mRNA abundances of insulin-like growth factor (IGF)-I and its receptor, and key steroidogenic enzymes responsible for glucocorticoid synthesis. The relative mass of the adrenal medulla and its proportion of the adrenal gland were increased by thyroid hormone deficiency in utero, without any change in expression of phenylethanolamine N-methyltransferase or the IGF system. Conclusions: Thyroid hormones are important regulators of the structure and secretory capacity of the pituitary-adrenal axis before birth. In hypothyroid fetuses, low plasma cortisol may be due to impaired adrenocortical growth and steroidogenic enzyme expression, secondary to low circulating ACTH concentration. Greater corticotroph population in the anterior pituitary gland of the hypothyroid fetus indicates compensatory cell proliferation and that there may be abnormal corticotroph capacity for ACTH synthesis and/or impaired hypothalamic input. Suppression of the development of the fetal HPA axis by thyroid hormone deficiency may contribute to the delay in fetal maturation and delivery observed in hypothyroid offspring.

Neonatal glucocorticoid overexposure alters cardiovascular function in young adult horses in a sex-linked manner.

Prenatal glucocorticoid overexposure has been shown to programme adult cardiovascular function in a range of species, but much less is known about the long-term effects of neonatal glucocorticoid overexposure. In horses, prenatal maturation of the hypothalamus-pituitary-adrenal axis and the normal prepartum surge in fetal cortisol occur late in gestation compared to other precocious species. Cortisol levels continue to rise in the hours after birth of full-term foals and increase further in the subsequent days in premature, dysmature and maladapted foals. Thus, this study examined the adult cardiovascular consequences of neonatal cortisol overexposure induced by adrenocorticotropic hormone administration to full-term male and female pony foals. After catheterisation at 2-3 years of age, basal arterial blood pressures (BP) and heart rate were measured together with the responses to phenylephrine (PE) and sodium nitroprusside (SNP). These data were used to assess cardiac baroreflex sensitivity. Neonatal cortisol overexposure reduced both the pressor and bradycardic responses to PE in the young adult males, but not females. It also enhanced the initial hypotensive response to SNP, slowed recovery of BP after infusion and reduced the gain of the cardiac baroreflex in the females, but not males. Basal diastolic pressure and cardiac baroreflex sensitivity also differed with sex, irrespective of neonatal treatment. The results show that there is a window of susceptibility for glucocorticoid programming during the immediate neonatal period that alters cardiovascular function in young adult horses in a sex-linked manner.

Placental glycogen stores and fetal growth: insights from genetic mouse models.

The placenta performs a range of crucial functions that support fetal growth during pregnancy, including facilitating the supply of oxygen and nutrients to the fetus, removal of waste products from the fetus and the endocrine modulation of maternal physiology. The placenta also stores glucose in the form of glycogen, the function of which remains unknown. Aberrant placental glycogen storage in humans is associated with maternal diabetes during pregnancy and pre-eclampsia, thus linking placental glycogen storage and metabolism to pathological pregnancies. To understand the role of placental glycogen in normal and complicated pregnancies, we must turn to animal models. Over 40 targeted mutations in mice demonstrate the defects in placental cells that store glycogen and suggest that placental glycogen represents a source of readily mobilized glucose required during periods of high fetal demand. However, direct functional evidence is currently lacking. Here, we evaluate these genetic mouse models with placental phenotypes that implicate glycogen trophoblast cell differentiation and function to illuminate the common molecular pathways that emerge and to better understand the relationship between placental glycogen and fetal growth. We highlight the current limitations in exploring the key questions regarding placental glycogen storage and metabolism and define how to experimentally overcome these constraints.

Thyroid Deficiency Before Birth Alters the Adipose Transcriptome to Promote Overgrowth of White Adipose Tissue and Impair Thermogenic Capacity.

Background: Development of adipose tissue before birth is essential for energy storage and thermoregulation in the neonate and for cardiometabolic health in later life. Thyroid hormones are important regulators of growth and maturation in fetal tissues. Offspring hypothyroid in utero are poorly adapted to regulate body temperature at birth and are at risk of becoming obese and insulin resistant in childhood. The mechanisms by which thyroid hormones regulate the growth and development of adipose tissue in the fetus, however, are unclear. Methods: This study examined the structure, transcriptome, and protein expression of perirenal adipose tissue (PAT) in a fetal sheep model of thyroid hormone deficiency during late gestation. Proportions of unilocular (UL) (white) and multilocular (ML) (brown) adipocytes, and UL adipocyte size, were assessed by histological and stereological techniques. Changes to the adipose transcriptome were investigated by RNA sequencing and bioinformatic analysis, and proteins of interest were quantified by Western blotting. Results: Hypothyroidism in utero resulted in elevated plasma insulin and leptin concentrations and overgrowth of PAT in the fetus, specifically due to hyperplasia and hypertrophy of UL adipocytes with no change in ML adipocyte mass. RNA sequencing and genomic analyses showed that thyroid deficiency affected 34% of the genes identified in fetal adipose tissue. Enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) pathways were associated with adipogenic, metabolic, and thermoregulatory processes, insulin resistance, and a range of endocrine and adipocytokine signaling pathways. Adipose protein levels of signaling molecules, including phosphorylated S6-kinase (pS6K), glucose transporter isoform 4 (GLUT4), and peroxisome proliferator-activated receptor γ (PPARγ), were increased by fetal hypothyroidism. Fetal thyroid deficiency decreased uncoupling protein 1 (UCP1) protein and mRNA content, and UCP1 thermogenic capacity without any change in ML adipocyte mass. Conclusions: Growth and development of adipose tissue before birth is sensitive to thyroid hormone status in utero. Changes to the adipose transcriptome and phenotype observed in the hypothyroid fetus may have consequences for neonatal survival and the risk of obesity and metabolic dysfunction in later life.