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Key points The prevalence of obesity and non-alcoholic fatty liver disease in children is dramatically increasing at the same time as consumption of foods with a high sugar content. Intake of high fructose corn syrup (HFCS) is a possible etiology as it is thought to be more lipogenic than glucose. In a mouse model, HFCS intake during adolescence increased fat mass and hepatic lipid levels in male and female mice. However, only males showed impaired glucose tolerance. Multiple metabolites including lipids, bile acids, carbohydrates, and amino acids were altered in liver in a sex-specific manner at 6 weeks of age. Some of these changes were also present in adulthood even though HFCS exposure ended at 6 weeks. HFCS significantly altered the gut microbiome which was associated with changes in key microbial metabolites. These results suggest that HFCS intake during adolescence has profound metabolic changes that are linked to changes in the microbiome and these changes are sex-specific. Abstract The rapid increase in obesity, diabetes and fatty liver disease in children over the past 20 years has been linked to increased high fructose corn syrup (HFCS) consumption making it essential to determine the short and long-term effects of HFCS during this vulnerable developmental window. We hypothesized that HFCS exposure during adolescence significantly impairs hepatic metabolic signaling pathways and alters gut microbial composition contributing to changes in energy metabolism with sex-specific effects. C57bl/6J mice with free access to HFCS during adolescence (3-6 weeks of age) underwent glucose tolerance and body composition testing and hepatic metabolomics, gene expression and triglyceride content analysis at 6 and 30 weeks of age (n = 6-8 per sex). At 6 weeks HFCS-exposed mice had significant increases in fat mass, glucose intolerance, hepatic triglycerides (females) and de novo lipogenesis gene expression (ACC, DGAT, FAS, ChREBP, SCD, SREBP, CPT and PPARα) with sex-specific effects. At 30 weeks HFCS-exposed mice also had abnormalities in glucose tolerance (males) and fat mass (females). HFCS exposure enriched carbohydrate, amino acid, long chain fatty acid and secondary bile acid metabolism at 6 weeks with changes in secondary bile metabolism at 6 and 30 weeks. Microbiome studies performed immediately before and after HFCS exposure identified profound shifts of microbial species in male mice only. In summary, a short-term HFCS exposure during adolescence induces fatty liver, alters important metabolic pathways, some of which continue to be altered in adulthood, and changes the microbiome in a sex-specific manner. This article is protected by copyright. All rights reserved.

The placenta is a metabolically active interfacial organ that plays crucial roles in fetal nutrient delivery, gas exchange and waste removal reflecting dynamic maternal and fetal interactions during gestation. There is growing evidence that the sex of the placenta influences fetal responses to external stimuli in utero, such as changes in maternal nutrition and exposure to environmental stressors. However, the exact biochemical mechanisms associated with sex-specific metabolic adaptations during pregnancy and its link to placental function and fetal development remain poorly understood. Herein, multisegment injection-capillary electrophoresis-mass spectrometry is used as a high throughput metabolomics platform to characterize lyophilized placental tissue (~2 mg dried weight) from C57BL/6J mice fed a standardized diet. Over 130 authentic metabolites were consistently measured from placental extracts when using a nontargeted metabolomics workflow with stringent quality control and robust batch correction. Our work revealed distinct metabolic phenotype differences that exist between male (n = 14) and female (n = 14) placentae collected at embryonic day E18.5. Intracellular metabolites associated with fatty acid oxidation and purine degradation were found to be elevated in females as compared to male placentae (p 0.40), including uric acid, valerylcarnitine, hexanoylcarnitine, and 3-hydroxyhexanolycarnitine. This murine model sheds new insights into sex-specific differences in placental mitochondrial function and protective mechanisms against deleterious oxidative stress that may impact fetal growth and birth outcomes later in life.

Maternal undernutrition decreases sperm production in male offspring, possibly through insulin-like growth factor (IGF-I). To test this hypothesis, we fed pregnant Wistar rats ad libitum with a standard diet (CONTROL) or fed 50% of CONTROL intake, either throughout pregnancy (UNP), lactation (UNL, or both (UNPL). After weaning, male offspring (n = 10 per treatment) were fed a standard diet until postnatal day 160, when testes process for histological and molecular analyses. IGF-I immunostaining area and intensity in the testis were greater (P = 0.003) in the UNPL group compared to CONTROL, but lower in the UNP group (P < 0.0001). Levels of IGF-I receptor transcript were lower in the UNPL and UNL groups, compared to CONTROL. There were more Ki-67-positive germ and Sertoli cells, in all underfed groups than in CONTROL. Compared to CONTROL, frequency of spermatogenic cycle stage VII was lower in all underfed groups, and seminiferous tubule diameter was smaller in UNP and UNPL. Plasma FSH concentrations were greater in UNP male offspring compared to all groups (P = 0.05), whereas inhibin B concentrations were greater in UNP (P = 0.01) and UNL (P = 0.003) than in CONTROL or UNPL. Thus, prenatal undernutrition leads to a decrease in testicular IGF-I levels, whereas of pre- and postnatal undernutrition increased testicular IGF-I levels and decreased amounts of IGF-I receptor mRNA in adult offspring. We conclude that maternal undernutrition during pregnancy and lactation leads to long-lasting effects on adult male offspring testicular morphology, spermatogenesis, and IGF-I testicular system.

Paternal obesity predisposes offspring to metabolic dysfunction, but the underlying mechanisms remain unclear. We investigated whether this metabolic dysfunction is associated with changes in placental vascular development and is fueled by endoplasmic reticulum (ER) stress-mediated changes in fetal hepatic development. We also determined whether paternal obesity indirectly affects the in utero environment by disrupting maternal metabolic adaptations to pregnancy. Male mice fed a standard chow or high fat diet (60%kcal fat) for 8–10 weeks were time-mated with female mice to generate pregnancies and offspring. Glucose tolerance was evaluated in dams at mid-gestation (embryonic day (E) 14.5) and late gestation (E18.5). Hypoxia, angiogenesis, endocrine function, macronutrient transport, and ER stress markers were evaluated in E14.5 and E18.5 placentae and/or fetal livers. Maternal glucose tolerance was assessed at E14.5 and E18.5. Metabolic parameters were assessed in offspring at ~60 days of age. Paternal obesity did not alter maternal glucose tolerance but induced placental hypoxia and altered placental angiogenic markers, with the most pronounced effects in female placentae. Paternal obesity increased ER stress-related protein levels (ATF6 and PERK) in the fetal liver and altered hepatic expression of gluconeogenic factors at E18.5. Offspring of obese fathers were glucose intolerant and had impaired whole-body energy metabolism, with more pronounced effects in female offspring. Metabolic deficits in offspring due to paternal obesity may be mediated by sex-specific changes in placental vessel structure and integrity that contribute to placental hypoxia and may lead to poor fetal oxygenation and impairments in fetal metabolic signaling pathways in the liver.

Objective:Maternal obesity is associated with obesity and metabolic disorders in offspring. However, there remains a paucity of data on strategies to reverse the effects of maternal obesity on maternal and offspring health. With maternal undernutrition, taurine supplementation improves outcomes in offspring mediated in part via improved glucose–insulin homeostasis. The efficacy of taurine supplementation in the setting of maternal obesity on health and well-being of offspring is unknown. We examined the effects of taurine supplementation on outcomes related to growth and metabolism in offspring in a rat model of maternal obesity.Design:Wistar rats were randomised to: 1) control diet during pregnancy and lactation (CON); 2) CON with 1.5% taurine in drinking water (CT); 3) maternal obesogenic diet (MO); or 4) MO with taurine (MOT). Offspring were weaned onto the control diet for the remainder of the study.Results:At day 150, offspring body weights and adipose tissue weights were increased in MO groups compared to CON. Adipose tissue weights were reduced in MOT versus MO males but not females. Plasma fasting leptin and insulin were increased in MO offspring groups but were not altered by maternal taurine supplementation. Plasma homocysteine concentrations were reduced in all maternal taurine-supplemented offspring groups. There were significant interactions across maternal diet, taurine supplementation and sex for response to an oral glucose tolerance test , a high-fat dietary preference test and pubertal onset in offspring.Conclusions:These results demonstrate that maternal taurine supplementation can partially ameliorate adverse developmental programming effects in offspring in a sex-specific manner.

We investigated whether diet-induced changes in the maternal intestinal microbiota were associated with changes in bacterial metabolites and their receptors, intestinal inflammation, and placental inflammation at mid-gestation (E14.5) in female mice fed a control (17% kcal fat, n = 7) or a high-fat diet (HFD 60% kcal fat, n = 9; ad libitum) before and during pregnancy. Maternal diet-induced obesity (mDIO) resulted in a reduction in maternal fecal short-chain fatty acid producing Lachnospiraceae, lower cecal butyrate, intestinal antimicrobial peptide levels, and intestinal SCFA receptor Ffar3, Ffar2 and Hcar2 transcript levels. mDIO increased maternal intestinal pro-inflammatory NFκB activity, colonic CD3+ T cell number, and placental inflammation. Maternal obesity was associated with placental hypoxia, increased angiogenesis, and increased transcript levels of glucose and amino acid transporters. Maternal and fetal markers of gluconeogenic capacity were decreased in pregnancies complicated by obesity. We show that mDIO impairs bacterial metabolite signaling pathways in the mother at mid-gestation, which was associated with significant structural changes in placental blood vessels, likely as a result of placental hypoxia. It is likely that maternal intestinal changes contribute to adverse maternal and placental adaptations that, via alterations in fetal hepatic glucose handling, may impart increased risk of metabolic dysfunction in offspring.

Chronic low-grade systemic inflammation in obesity contributes to the development and progression of aspects of metabolic syndrome. In obese male mice, expanded adipose tissue releases proinflammatory cytokines, including TNF, which promotes an increase in immature, proinflammatory, peripheral blood Ly-6Chigh monocytes. The aim of this study was to characterize how TNF alters circulating cellular immunity in female mice with diet-induced obesity. We initially quantified peripheral blood immune cells by flow cytometry in female wild-type C57BL/6J mice after 3–30 wk of allocation to a high-fat (HF) or standard chow diet. We assessed effects of diet and time on neutrophil, monocyte, B cell, NK cell, CD4+ T cell, and CD8+ T cell populations. There was a significant interaction of the effects of diet type and time on the numbers and prevalence of circulating total monocytes and Ly-6Chigh, Ly-6Clow, and Ly-6C− subsets. Circulating monocytes, in particular Ly-6Chigh monocytes, were increased in HF-fed mice compared with chow-fed mice. Ly-6Chigh monocytes from HF-fed mice also had a more immature phenotype yet were highly responsive to the chemotactic ligand CCL2 and had greater intracellular production of TNF. Comparisons of the effects of HF diet feeding in littermate wild-type (TNF+/+) and TNF−/− female mice showed that genetic ablation of TNF did not protect from higher adiposity or an increase in circulating, immature, proinflammatory Ly-6Chigh monocytes during HF diet–induced obesity. These data emphasize the importance of considering biological sex when determining the mechanisms of TNF action in obesity-induced cellular inflammation and in other chronic inflammatory conditions.

We tested whether changes in Sertoli cell transcription factors and germ cell heat shock proteins (HSPs) are linked to the effects of maternal undernutrition on male offspring fertility. Rats were fed ad libitum with a standard diet (CONTROL) throughout pregnancy and lactation or with 50% of CONTROL intake throughout pregnancy (UNP) or lactation (UNL) or both periods (UNPL). After postnatal Day 21, 10 male pups per group were fed a standard diet ad libitum until postnatal Day 160 when testes were processed for histological, mRNA and immunohistochemical analyses. Compared with CONTROL: caspase-3 was increased in UNP and UNPL (P = 0.001); Bax was increased in UNL (P = 0.002); Bcl-2 (P

Key points Traditionally the female sex, compared with the male sex, has been perceived as having greater variability in many physiological traits, including within the immune system. We investigated effects of biological sex and the female reproductive cycle on numbers of circulating leukocytes in C57BL/6J mice. We show that biological sex, but not female reproductive cyclicity, has a significant effect on peripheral blood immune cell prevalence and variability, and that sex differences were not consistent amongst common inbred laboratory mouse strains. We found that male C57BL/6J mice, compared with female mice, have greater variability in peripheral blood immunophenotype, and that this was influenced by body weight. We created summary tables for researchers to facilitate experiment planning and sample size calculations for peripheral immune cells that consider the effects of biological sex. Abstract Immunophenotyping (i.e. quantifying the number and types of circulating leukocytes) is used to characterize immune changes during health and disease, and in response to pharmacological and other interventions. Despite the importance of biological sex in immune function, there is considerable uncertainty amongst researchers as to the extent to which biological sex or the female reproductive cycle influence blood immunophenotype. We quantified circulating leukocytes by multicolour flow cytometry in young C57BL/6J mice and assessed the effects of the reproductive cycle, biological sex, and other experimental and biological factors on data variability. We found that there are no significant effects of the female reproductive cycle on the prevalence of peripheral blood B cells, NK cells, CD4+ T cells, CD8+ T cells, monocytes, or neutrophils. Immunophenotype composition and variability do not significantly change between stages of the female reproductive cycle. There are, however, sex-specific differences in immune cell prevalence, with fewer monocytes, neutrophils, and NK cells in female mice. Surprisingly, immunophenotype is more variable in male mice, and weight is a significant contributing factor. We provide tools for researchers to perform a priori sample size calculations for two-group and factorial analyses. We show that immunophenotype varies between inbred mouse strains, and that using equal sample sizes of male and female mice is not always appropriate for within-sex evaluations of immune cell populations in peripheral blood.

Objective Obesity and diabetes increase circulating levels of microbial components derived from the gut microbiota. Individual bacterial factors (i.e., postbiotics) can have opposing effects on blood glucose. Methods We tested the net effect of gut bacterial extracts on blood glucose in mice using a microbiota-based vaccination strategy. Results Male and female mice had improved glucose and insulin tolerance five weeks after a single subcutaneous injection of a specific dose of a bacterial extract obtained from the luminal contents of the upper small intestine (SI), lower SI, or cecum. Injection of mice with intestinal extracts from germ-free mice revealed that bacteria were required for a microbiota-based vaccination to improve blood glucose control. Vaccination of Nod1−/−, Nod2−/−, and Ripk2−/− mice showed that each of these innate immune proteins was required for bacterial extract injection to improve blood glucose control. A microbiota-based vaccination promoted an immunoglobulin-G (IgG) response directed against bacterial extract antigens, where subcutaneous injection of mice with the luminal contents of the lower SI elicited a bacterial extract-specific IgG response that is compartmentalized to the lower SI of vaccinated mice. A microbiota-based vaccination was associated with an altered microbiota composition in the lower SI and colon of mice. Lean mice only required a single injection of small intestinal-derived bacterial extract, but high fat diet (HFD)-fed, obese mice required prime-boost bacterial extract injections for improvements in blood glucose control. Conclusions Subversion of the gut barrier by vaccination with a microbiota-based extract engages innate immunity to promote long-lasting improvements in blood glucose control in a dose-dependent manner., Graphical abstract Image 1, Highlights • Subcutaneous injection of gut bacterial extracts improved blood glucose control in mice. • Microbiota-based vaccination engaged NOD1-NOD2-RIPK2 to alter blood glucose. • Microbiota-based vaccination promoted a proximal gut IgG response. • Microbiota-based vaccination altered the composition of the gut microbiome. • Obese mice required prime-boost injections to improve blood glucose control.

Histological examination of the rat placenta and fetus is uncommon. Toxicological studies mainly rely on gross examination of the fetus and on fetal and placental weights. These are often insufficient to assess the fetal and placental toxicity of xenobiotics. The small size of the fetus makes its dissection labor-intensive. Thus, our objective was to develop a simple and accurate technique to evaluate the rat fetus and placenta. Sprague-Dawley rat fetuses at gestational day 19.5 ( n = 18) and their placentas ( n = 32) were fixed in formalin. Placentas were cut transversally in the center. Fetuses were cut following a freehand whole-body serial sectioning diagram adapted from Wilson’s method. Sections were stained with hematoxylin–eosin–phloxine–saffron, and histomorphometry was used to measure the area of the fetal placental region (27.2 ± 1.7 mm2), including the labyrinth (22.2 ± 1.0 mm2) and the basal zone (4.8 ± 0.8 mm2). Our whole-fetus serial sectioning technique resulted in 12 precise cutting planes that fit on 3 histological slides, enabling the examination of most organs without labor-intensive dissection. Quantitative analysis of placental areas improves the understanding of the pathogenesis of treatment-related changes. This technique provides a standardized method for future research in pertinent fields such as developmental biology and toxicology.

It is well established that early life environmental signals, including nutrition, set the stage for long-term health and disease risk – effects that span multiple generations. This relationship begins early, in the periconceptional period and extends into embryonic, fetal and early infant phases of life. Now known as the Developmental Origins of Health and Disease (DOHaD), this concept describes the adaptations that a developing organism makes in response to early life cues, resulting in adjustments in homeostatic systems that may prove maladaptive in postnatal life, leading to an increased risk of chronic disease and/or the inheritance of risk factors across generations. Reproductive maturation and function is similarly influenced by early life events. This should not be surprising, since primordial germ cells are established early in life and thus vulnerable to early life adversity. A multitude of ‘modifying’ cues inducing developmental adaptations have been identified that result in changes in reproductive development and impairments in reproductive function. Many types of nutritional challenges including caloric restriction, macronutrient excess and micronutrient insufficiencies have been shown to induce early life adaptations that produce long-term reproductive dysfunction. Many pathways have been suggested to underpin these associations, including epigenetic reprogramming of germ cells. While the mechanisms still remain to be fully investigated, it is clear that a lifecourse approach to understanding lifetime reproductive function is necessary. Furthermore, investigations of the impacts of early life adversity must be extended to include the paternal environment, especially in epidemiological and clinical studies of offspring reproductive function.

Background: The mechanisms mediating the impacts of fetal growth restriction (FGR) on follicular development are commonly studied in mouse/rat models, where ovarian development occurs largely during the early postnatal period. These models have shown that FGR is associated with premature follicle loss, early pubertal onset, and accelerated ovarian aging. Whether the same occurs in precocious species is unknown. Objective: Since guinea pig follicle development occurs in utero in a manner consistent with human ovarian development, we sought to determine whether FGR had similar impacts on guinea pig ovarian development. Methods: Dunkin-Hartley guinea pig dams were randomized to receive a control (CON) or a nutrient-restricted diet (FGR) prior to conception until weaning. Offspring ovaries were collected at prepubertal (postnatal day [P] 25) and young adult (P110) time points. Results: Prepubertal offspring exposed to FGR showed little differences in ovarian transcript levels and follicle counts. Young adult FGR offspring, however, showed reductions in the number of transitioning, primary, and antral follicles, as well as corpora lutea. This loss in follicles was associated with reduced insulin-like growth factor receptor and growth differentiation factor-9 messenger RNA levels in FGR P110 offspring compared to CON. Conclusion: We demonstrate that FGR in guinea pigs is accompanied by perturbations in signaling pathways essential for proper follicle growth and manifests as reductions in growing follicles in offspring, but these changes do not manifest until postpuberty. These data support the fact that accelerated reproductive maturation/aging is a conserved phenotype that is associated with in utero nutritional adversity.

Acephate has been used extensively as an insecticide in agriculture. Its downstream sequelae are associated with hyperglycemia, lipid metabolism dysfunction, DNA damage, and cancer, which are rapidly growing epidemics and which lead to increased morbidity and mortality rates and soaring health-care costs. Developing interventions will require a comprehensive understanding of which excess insecticides during perinatal life can cause insulin resistance and type 2 diabetes. A Wistar rat animal model suggests that acephate exposure during pregnancy and lactation causes alterations in maternal glucose metabolism and programs the offspring to be susceptible to type 2 diabetes at adulthood. Therapeutic approaches based on preventive actions to food contaminated with insecticides during pregnancy and lactation could prevent new cases of type 2 diabetes.

Prenatal glucocorticoids, commonly used in women at risk of preterm delivery, can predispose the newborn to disease in later life. Since male reproductive function is likely to reflect testis development during fetal life, we studied the effects of prenatal glucocorticoids on two key intra-testicular factors that play roles in cellular proliferation and differentiation, 3β-hydroxysteroid dehydrogenase (3β-HSD) and inhibin-α. Pregnant sheep (n=42) were treated with betamethasone (0.5 mg/kg) or saline (control) at 104, 111 and 118 days of gestation (DG). Testicular tissue was sampled from fetuses at 121 and 132DG, and from lambs at 45 and 90 postnatal days (PD). Within the betamethasone treated group, 3β-HSD immunostaining area was greater at 121DG than at 90PD (P=0.04), but the intensity of immunostaining was higher at 90PD than at 121DG (P=0.04), 132DG (P=0.04) and 45PD (P=0.03). Control animals showed no changes in 3β-HSD area or intensity of immunostaining. No significant differences were observed between treated and control animals in immunostaining area, but immunostaining was more intense in the treated group than in the control group at 90PD (P=0.03). For inhibin-α, the proportion of immunostaining area declined in treated offspring from 121DG to 45PD, in contrast to control values, but recovered fully by 90PD, concomitantly with the onset of spermatogenesis. In conclusion, prenatal betamethasone increased the postnatal testicular expression of inhibin-α but reduced the expression of 3β-HSD. These effects could compromise androgen-mediated testicular development and therefore adult capacity for spermatogenesis.

Inflammation contributes to obesity‐related hyperinsulinemia and insulin resistance, which often precede type 2 diabetes. Inflammation is one way that obesity can promote insulin resistance. It is not clear if the extent of obesity, hyperinsulinemia, or hyperglycemia, underpins changes in cellular immunity during diet‐induced obesity. In particular, the requirement for obesity or directionality in the relationship between insulin resistance and monocyte characteristics is poorly defined. Inflammatory cytokines such as tumor necrosis factor (TNF) can contribute to insulin resistance. It is unclear if TNF alters monocytosis or specific markers of cellular immunity in the context of obesity. We measured bone marrow and blood monocyte characteristics in WT and TNF −/− mice that were fed obesogenic, high fat (HF) diets. We also used hyperglycemic Akita mice and mice implanted with insulin pellets in order to determine if glucose or insulin were sufficient to alter monocyte characteristics. We found that diet‐induced obesity in male mice increased the total number of monocytes in blood, but not in bone marrow. Immature, inflammatory (Ly6Chigh) monocytes decreased within the bone marrow and increased within peripheral blood of HF‐fed mice. We found that neither hyperinsulinemia nor hyperglycemia was sufficient to induce the observed changes in circulating monocytes in the absence of diet‐induced obesity. In obese HF‐fed mice, antibiotic treatment lowered insulin and insulin resistance, but did not alter circulating monocyte characteristics. Fewer Ly6Chigh monocytes were present within the blood of HF‐fed TNF −/− mice in comparison to HF‐fed wild‐type (WT) mice. The prevalence of immature Ly6Chigh monocytes in the blood correlated with serum insulin and insulin resistance irrespective of the magnitude of adipocyte or adipose tissue hypertrophy in obese mice. These data suggest that diet‐induced obesity instigates a TNF‐dependent increase in circulating inflammatory monocytes, which predicts increased blood insulin and insulin resistance independently from markers of adiposity or adipose tissue expansion.

Key points Maternal obesity has been associated with shifts in intestinal microbiota, which may contribute to impaired barrier function Impaired barrier function may expose the placenta and fetus to pro-inflammatory mediators We investigated the impacts of diet-induced obesity in mice on maternal and fetal intestinal structure and placental vascularization Diet-induced obesity decreased maternal intestinal short chain fatty acids and their receptors, impaired gut barrier integrity and was associated with fetal intestinal inflammation. Placenta from obese mothers showed blood vessel immaturity, hypoxia, increased transcript levels of inflammation, autophagy and altered levels of endoplasmic reticulum stress markers. These data suggest that maternal intestinal changes probably contribute to adverse placental adaptations and also impart an increased risk of obesity in the offspring via alterations in fetal gut development. Abstract Shifts in maternal intestinal microbiota have been implicated in metabolic adaptations to pregnancy. In the present study, we generated cohorts of female C57BL/6J mice fed a control (17% kcal fat, n = 10-14) or a high-fat diet (HFD 60% kcal from fat, n = 10-14; ad libitum) aiming to investigate the impact on the maternal gut microbiota, intestinal inflammation and gut barrier integrity, placental inflammation and fetal intestinal development at embryonic day 18.5. HFD was associated with decreased relative abundances of short-chain fatty acid (SCFA) producing genera during pregnancy. These diet-induced shifts paralleled decreased maternal intestinal mRNA levels of SCFA receptor Gpr41, modestly decreased cecal butyrate, and altered mRNA levels of inflammatory cytokines and immune cell markers in the maternal intestine. Maternal HFD resulted in impaired gut barrier integrity, with corresponding increases in circulating maternal levels of lipopolysaccharide (LPS) and tumour necrosis factor. Placentas from HFD dams demonstrated blood vessel immaturity and hypoxia; decreased free carnitine, acylcarnitine derivatives and trimethylamine-N-oxide; and altered mRNA levels of inflammation, autophagy, and ER stress markers. HFD exposed fetuses had increased activation of nuclear factor-kappa B and inhibition of the unfolded protein response in the developing intestine. Taken together, these data suggest that HFD intake prior to and during pregnancy shifts the composition of the maternal gut microbiota and impairs gut barrier integrity, resulting in increased maternal circulating LPS, which may ultimate contribute to changes in placental vascularization and fetal gut development.

Reproductive abnormalities are included as health complications in offspring exposed to poor prenatal nutrition. We have previously shown in a rodent model that offspring born to nutrient restriction during pregnancy are born small, enter puberty early, and display characteristics of early ovarian aging as adults. The present study investigated whether key proteins involved in follicle recruitment and growth mediate ovarian follicle loss. Pregnant rats were randomized to a standard diet throughout pregnancy and lactation (CON), or a calorie-restricted (50% of control) diet (UN) during pregnancy. Offspring reproductive phenotype was investigated at postnatal days 4, 27, and 65. Maternal UN resulted in young adult (P65) irregular estrous cyclicity due to persistent estrus, a significant loss of antral follicles, corpora lutea, and an increase in atretic follicles. This decrease in growing follicles in UN offspring appears to be due to increased apoptosis as seen by immunopositive staining of pro-apoptotic factor CASP3 (caspase 3) in ovaries of young adult offspring. UN prepubertal offspring had reduced expression levels of Fshr in antral follicles, which may contribute to a decrease in PI3K/AKT activation evident as a decrease in pAKT immunolocalization in prepubertal antral follicles. Moreover, neonatal ovaries of UN offspring show decreased levels of immunopositive staining for AMHR2 (anti-mullerian hormone receptor 2). Collectively, these data demonstrate that maternal UN during pregnancy impacts ovarian function in offspring as early as P65 and provides a model for understanding the mechanisms driving early life UN-induced follicle loss and reproductive dysfunction.

Pregnancy is accompanied by maternal physiological adaptations including metabolic, endocrine, immune, cardiovascular, skeletomuscular and neurological modifications that facilitate fetal and placental growth and development. Emerging evidence suggests that the maternal intestinal microbiota is modified over the course of healthy pregnancy. We have recently identified a maternal intestinal microbial shift within hours of conception; a shift that continued with advancing gestation. It is possible that maternal gut bacterial profiles might be associated with the known endocrine changes that accompany the female reproductive (estrous) cycle. Methods: To determine whether the estrous cycle influenced the shifts in the maternal intestinal microbiota, time-matched fecal pellets were collected daily for 3 consecutive estrous cycles from individually housed, non-pregnant female C57BL/6J mice (n = 10) fed a control diet. Estrous stage was identified by cell type predominance in vaginal cytological samples. The corresponding fecal pellets for each estrous stage were processed for bacterial 16S rRNA sequencing of the variable 3 (V3) region. Results: Estrous cycle stage accounted for a very small and not statistically significant proportion of the variation in the fecal microbiota according to PERMANOVA testing performed on Bray-Curtis dissimilarity scores. These values displayed no significant clustering of fecal microbial communities by estrous stage. Conclusion: The estrous cycle does not result in any significant shift in the intestinal microbial community in the reproductively mature, regularly cycling female mouse.

Fructose is an increasingly common constituent of the Westernized diet due to cost and production efficiencies. Although an integral component of our pre-industrial revolution diet, over the past two decades human and animal studies have highlighted that excessive fructose intake appears to be associated with adverse metabolic effects. Excessive intake of fructose is the combined result of increased total energy consumption and increased portion sizes of foods, which often incorporate the fructose-containing sugars sucrose and high-fructose corn-syrup (HFCS). The adverse metabolic effects following excessive fructose consumption have become a hot topic in mainstream media and there is now rigorous scientific debate regarding periods of exposure, dosage levels, interactive effects with other sugars and fats and mechanisms underlying the actions of fructose. There is still a degree of controversy regarding the extent to which sugars such as sucrose and HFCS have contributed to the current epidemic of obesity and diabetes. Furthermore, an increasing number of infants are being exposed to sugar-sweetened food and beverages before birth and during early postnatal life, highlighting the importance of determining the long-term effects of this perinatal exposure on the developing offspring. There are limited human observational and controlled studies identifying associations of excessive sweetened food and beverage consumption with poor pregnancy outcomes. Animal research has demonstrated an increased incidence of gestational diabetes as well as altered maternal, fetal and offspring metabolic function, although the long-term effects and the mechanism underlying these perturbations are ill defined. This review aims to understand the role of early life fructose exposure in modifying postnatal risk of disease in the offspring, focusing on fructose intake during pregnancy and in early postnatal life.

It is well established that altered maternal nutrition may induce long-term metabolic consequences in offspring. However, the effects of maternal undernutrition during different developmental windows on sex-specific growth and metabolism in offspring are not well defined. We investigated the effect of moderate maternal undernutrition during pregnancy and/or lactation on postnatal growth and metabolic outcomes in offspring. Wistar rats were randomly assigned to one of four groups: (1) control (CONT) dams fed a standard diet throughout pregnancy and lactation; (2) dams undernourished to 50 % of CONT during pregnancy (UNP); (3) dams fed at 50 % of CONT throughout lactation (UNL); (4) dams fed at 50 % of CONT throughout pregnancy and lactation (UNPL). UNP and UNPL offspring were lighter at birth compared to CONT and UNL. UNL and UNPL offspring were growth restricted at weaning and remained smaller into adulthood. UNP males and females developed increased adiposity and hyperleptinaemia in adulthood compared to all other groups. Adiposity in UNL and UNPL males was similar to CONT offspring. In UNL and UNPL females, adiposity was lower than for CONT females. Markers of bone mass, lipid metabolism and hepatic function were altered in UNP offspring but were similar in UNL and UNPL offspring compared to CONT. Lack of catch-up growth during lactation in offspring of undernourished mothers prevented development of adiposity and related metabolic disorders in later life. These data highlight that the timing and duration of undernutrition during critical windows of development exert differential effects on postnatal outcomes in a sex-specific manner.

The incidence of obesity and overweight has reached epidemic proportions in the developed world as well as in those countries transitioning to first world economies, and this represents a major global health problem. Concern is rising over the rapid increases in childhood obesity and metabolic disease that will translate into later adult obesity. Although an obesogenic nutritional environment and increasingly sedentary lifestyle contribute to our risk of developing obesity, a growing body of evidence links early life nutritional adversity to the development of long-term metabolic disorders. In particular, the increasing prevalence of maternal obesity and excess maternal weight gain has been associated with a heightened risk of obesity development in offspring in addition to an increased risk of pregnancy-related complications. The mechanisms that link maternal obesity to obesity in offspring and the level of gene-environment interactions are not well understood, but the early life environment may represent a critical window for which intervention strategies could be developed to curb the current obesity epidemic. This paper will discuss the various animal models of maternal overnutrition and their importance in our understanding of the mechanisms underlying altered obesity risk in offspring.

Fetal exposure to elevated levels of bioactive glucocorticoids early in gestation, as in suspected cases of congenital adrenal hyperplasia, may result in adverse neurological events. Fetal hypothalamic-pituitary-adrenal development and function may be involved. We investigated immediate and long-term effects of maternal dexamethasone (DEX) administration early in pregnancy on fetal growth and pituitary-adrenal activity in sheep. Pregnant ewes carrying singleton fetuses (total n = 119) were randomized to control (2 ml saline/ewe) or DEX-treated groups (im injections of 0.14 mg/kg ewe weight · 12 h) at 40–41 d gestation (dG). At 50, 100, 125, and 140 dG, fetal plasma and tissues were collected. DEX-exposed fetuses were lighter than controls at 100 dG (P < 0.05) but not at any other times. Fetal plasma ACTH levels and pituitary POMC and PC-1 mRNA levels were similar between groups. Fetal plasma cortisol levels were significantly reduced after DEX exposure in both male and female fetuses at 50 dG (P < 0.05), were similar at 100 and 125 dG, but were significantly higher than controls at 140 dG. At 140 dG, there was increased adrenal P450C17 and 3β-HSD mRNA in female fetuses and reduced expression of ACTH-R mRNA in males. Fetal hepatic CBG mRNA levels mimicked plasma cortisol patterns. DEX exposure reduced CBG only in males at 50 dG (P < 0.05). Placental mRNA levels of 11β-HSD2 were increased after DEX in males (P < 0.05). Therefore, in sheep, early DEX may alter the developmental trajectory of the fetal hypothalamic-pituitary-adrenal axis, directly increasing fetal adrenal activation but not anterior pituitary function. In females, this effect may be attributed, in part, to increased fetal adrenal steroidogenic activity.

To determine the expression of glucocorticoid metabolizing and action genes in the hippocampus of fetal, neonatal, and adult sheep. Pregnant ewes (or their fetuses) received intramuscular injections of saline or betamethasone (BETA, 0-5 mg/kg) at 104, 111, 118, and/or 125 days of gestation (dG). Hippocampal tissue was collected prior to (75, 84, and 101 dG), during (109 and 116 dG), or after (121, 132, and 146 dG; 6 and 12 postnatal weeks; 3.5 years of age) saline or BETA injections. Hippocampal glucocorticoid receptor (GR), mineralocorticoid receptor (MR), and 11β-hydroxysteroid dehydrogenase (11βHSD)1 and 11βHSD2 mRNA levels were determined using qRT-PCR. Control animals late in gestation demonstrated a decrease in mRNA encoding GR and 11βHSD1, whereas 11βHSD2 was undetectable, consistent with a damping of the negative feedback influence of circulating or locally produced cortisol on the hypothalamic–pituitary–adrenal (HPA) axis. BETA-administration had transient effects on fetal GR and MR, and early in postnatal life (12 weeks of age) 11βHSD1 mRNA was increased. Hippocampal MR mRNA was elevated in adult offspring exposed to either one or four doses of maternal BETA (PP

OBJECTIVE: To determine the role of carotid sinus innervation on differential fetal organ growth during maternal nutrient restriction in late pregnancy. DESIGN: Randomised controlled study. SETTING: University research facility. SAMPLE: Thirty-nine Merino ewes. METHODS: At 113 days gestational age (dGA), fetuses were bilaterally carotid sinus denervated or sham denervated. From 118 dGA, the surgery groups were subdivided into two dietary groups, and their ewes were fed 100% of nutrient requirements or 50% until tissue collection at 140 dGA. This provided four groups (sham/control diet, sham/restricted diet, denervated/control diet and denervated/restricted diet). MAIN OUTCOME MEASURES: Fetal organ weights and hormone levels and maternal weight change during the dietary restriction. RESULTS: Adrenal glands were larger in sham/restricted diet fetuses than in sham/control diet or denervated/restricted diet fetuses (P < 0.05). Fetal adrenal weight and brain-to-liver weight ratio were positively related to maternal weight change during the nutritional challenge in sham fetuses only (P < 0.05). Fetal liver weight was negatively related to maternal weight change during nutritional challenge in sham fetuses only (P < 0.05). CONCLUSIONS: We have shown a reduction in liver growth but sparing of adrenal growth in response to moderate maternal undernutrition, which is dependent on intact carotid body innervation. This suggests a new role for the carotid bodies in the control of differential organ growth during such undernutrition

Aim Fructose consumption is associated with altered hepatic function and metabolic compromise and not surprisingly has become a focus for perinatal studies. We have previously shown that maternal fructose intake results in sex specific changes in fetal, placental and neonatal outcomes. In this follow-up study we investigated effects on maternal, fetal and neonatal hepatic fatty acid metabolism and immune modulation. Methods Pregnant rats were randomised to either control (CON) or high-fructose (FR) diets. Fructose was given in solution and comprised 20% of total caloric intake. Blood and liver samples were collected at embryonic day 21 (E21) and postnatal day (P)10. Maternal liver samples were also collected at E21 and P10. Liver triglyceride and glycogen content was measured with standard assays. Hepatic gene expression was measured with qPCR. Results Maternal fructose intake during pregnancy resulted in maternal hepatic ER stress, hepatocellular injury and increased levels of genes that favour lipogenesis. These changes were associated with a reduction in the NLRP3 inflammasome. Fetuses of mothers fed a high fructose diet displayed increased hepatic fructose transporter and reduced fructokinase mRNA levels and by 10 days of postnatal age, also have hepatic ER stress, and elevated IL1β mRNA levels. At P10, FR neonates demonstrated increased hepatic triglyceride content and particularly in males, associated changes in the expression of genes regulating beta oxidation and the NLRP3 inflammasome. Further, prenatal fructose results in sex-dependant changes in levels of key clock genes. Conclusions Maternal fructose intake results in age and sex-specific alterations in maternal fetal and neonatal free fatty acid metabolism, which may be associated in disruptions in core clock gene machinery. How these changes are associated with hepatic inflammatory processes is still unclear, although suppression of the hepatic inflammasome, as least in mothers and male neonates may point to impaired immune sensing.

The rise in the occurrence of obesity to epidemic proportions has made it a global concern. Great difficulty has been experienced in efforts to control this growing problem with lifestyle interventions. Thus, attention has been directed to understanding the events of one of the most critical periods of development, perinatal life. Early life adversity driven by maternal obesity has been associated with an increased risk of metabolic disease and obesity in the offspring later in life. Although a mechanistic link explaining the relationship between maternal and offspring obesity is still under investigation, the gut microbiota has come forth as a new factor that may play a role modulating metabolic function of both the mother and the offspring. Emerging evidence suggests that the gut microbiota plays a much larger role in mediating the risk of developing non-communicable disease, including obesity and metabolic dysfunction in adulthood. With the observation that the early life colonization of the neonatal and postnatal gut is mediated by the perinatal environment, the number of studies investigating early life gut microbial establishment continues to grow. This paper will review early life gut colonization in experimental animal models, concentrating on the role of the early life environment in offspring gut colonization and the ability of the gut microbiota to dictate risk of disease later in life.

Shifts in the maternal gut microbiome have been implicated in metabolic adaptations to pregnancy. We investigated how pregnancy and diet interact to influence the composition of the maternal gut microbiota. Female C57BL/6 mice were fed either a control or a high fat diet for 8 weeks prior to mating. After confirmation of pregnancy, maternal weight gain and food intake were recorded. Fecal pellets were collected at 2 timepoints prior to mating (at the beginning of the experiment, and after 6 weeks of the specified diet) and at 4 timepoints during pregnancy (gestation day 0.5, 5.5, 10.5, and 15.5). The microbial composition and predicted metabolic functionality of the non-pregnant and pregnant gut was determined via sequencing of the variable 3 region of the 16S rRNA gene. Upon conception, differences in gut microbial communities were observed in both control and high fat-fed mice, including an increase in mucin-degrading bacteria. Control versus high fat-fed pregnant mice possessed the most profound changes to their maternal gut microbiota as indicated by statistically significant taxonomic differences. High fat-fed pregnant mice, when compared to control-fed animals, were found to be significantly enriched in microbes involved in metabolic pathways favoring fatty acid, ketone, vitamin, and bile synthesis. We show that pregnancy-induced changes in the female gut microbiota occur immediately at the onset of pregnancy, are vulnerable to modulation by diet, but are not dependent upon increases in maternal weight gain during pregnancy. High fat diet intake before and during pregnancy results in distinctive shifts in the pregnant gut microbiota in a gestational-age dependent manner and these shifts predict significant differences in the abundance of genes that favor lipid metabolism, glycolysis and gluconeogenic metabolic pathways over the course of pregnancy.

Maternal obesity predisposes offspring to metabolic and reproductive dysfunction. We have shown previously that female rat offspring born to mothers fed a high-fat (HF) diet throughout pregnancy and lactation enter puberty early and display aberrant reproductive cyclicity. The mechanisms driving this reproductive phenotype are currently unknown thus we investigated whether changes in ovarian function were involved. Wistar rats were mated and randomized to: dams fed a control diet (CON) or dams fed a HF diet from conception until the end of lactation (HF). Ovaries were collected from fetuses at Embryonic Day (E) 20, and neonatal ovaries at Day 4 (P4), prepubertal ovaries at P27 and adult ovaries at P120. In a subset of offspring, the effects of a HF diet fed postweaning were evaluated. The present study shows that fetuses of mothers fed a HF diet had significantly fewer oocytes at E20, and in neonates, have reduced AMH signaling that may facilitate an increased number of assembled primordial follicles. Both prepubertally and in adulthood, ovaries show increased follicular atresia. As adults, offspring have reduced FSH responsiveness, low expression levels of estrogen receptor alpha (Eralpha), the oocyte-secreted factor, Gdf9, oocyte-specific RNA binding protein, Dazl, and high expression levels of the granulosa-cell derived factor, AMH, in antral follicles. Together, these data suggest that ovarian compromise in offspring born to HF-fed mothers may arise from changes already observable in the fetus and neonate and in the long term, associated with increased follicular atresia through adulthood.

Adults born preterm are at increased risk of impaired glucose tolerance and diabetes. Late gestation fetuses exposed to high blood glucose concentration also are at increased risk of impaired glucose tolerance as adults. Preterm babies commonly become hyperglycemic and are thus exposed to high blood glucose concentration at an equivalent stage of pancreatic maturation. It is not known whether preterm birth itself, or complications of prematurity, such as hyperglycemia, alter later pancreatic function. To distinguish these, we made singleton preterm lambs hyperglycemic (HYPER) for 12 days after birth with a dextrose infusion and compared them with vehicle-treated preterm and term controls and with HYPER lambs made normoglycemic with an insulin infusion. Preterm birth reduced β-cell mass, apparent by 4 weeks after term and persisting to adulthood (12 mo), and was associated with reduced insulin secretion at 4 months (juvenile) and reduced insulin mRNA expression in adulthood. Hyperglycemia in preterm lambs further down-regulated key pancreatic gene expression in adulthood. These findings indicate that reduced β-cell mass after preterm birth may be an important factor in increased risk of diabetes after preterm birth and may be exacerbated by postnatal hyperglycemia.

The effects of endogenous cortisol on binucleate cells (BNCs), which promote fetal growth, may be mediated by glucocorticoid receptors (GRs), and exposure to dexamethasone (DEX) in early pregnancy stages of placental development might modify this response. In this article, we have investigated the expression of GR as a determinant of these responses. Pregnant ewes carrying singleton fetuses (n = 119) were randomized to control (2 mL saline/ewe) or DEX-treated groups (intramuscular injections of 0.14 mg/kg ewe weight per 12 hours) at 40 to 41 days of gestation (dG). Placental tissue was collected at 50, 100, 125, and 140 dG. Total glucocorticoid receptor protein (GRt) was increased significantly by DEX at 50 and 125 dG in females only, but decreased in males at 125 dG as compared to controls. Glucocorticoid receptor α (GRα) protein was not changed after DEX treatment. Three BNC phenotypes were detected regarding GRα expression (++, +−, −−), DEX increased the proportion of (++) and decreased (−−) BNC at 140 dG. Effects were sex- and cell type dependent, modifying the responsiveness of the placenta to endogenous cortisol. We speculate that 3 maturational stages of BNCs exist and that the overall activity of BNCs is determined by the distribution of these 3 cell types, which may become altered through early pregnancy exposure to elevated glucocorticoids.

Up to 10% of pregnant women take antidepressants, of which selective serotonin reuptake inhibitors (SSRIs) are the most commonly prescribed. Using a rodent model, we investigated the reproductive impacts of perinatal SSRI treatment on reproductive cyclicity and function in female offspring.Virgin Wistar rats were given oral vehicle (n = 10) or fluoxetine hydrochloride (FLX, 10 mg/kg/d; n = 11) from 2 weeks prior to mating until weaning. Pubertal onset and reproductive cyclicity in offspring were assessed. Blood and ovarian tissues were collected for measures of reproductive function.Perinatal FLX tends to induce irregular reproductive cycles in adult offspring, which most commonly manifest as a prolonged estrus phase (FLX 34% vs control [CON] 10%) relative to CON offspring. The FLX offspring tended to have longer cycles (P = .052), had more secondary follicles (P = .0067), more total follicles (P = .0310), and increased apoptotic ovarian cells (P.001). Prenatally exposed FLX offspring demonstrated elevated ovarian messenger RNA (mRNA) levels of ERβ (P = .008), Cry1 (P = .043), and tryptophan hydroxylase 2 (P = .024), independent of stage of cycle. Ovarian mRNA levels of brain and muscle Arnt-like protein 1 (P = .046) and Pet-1 (P = .021) were increased in FLX offspring a manner that was reproductive cycle stage dependent.This is the first study to investigate the postnatal effects of maternal perinatal exposure to FLX on adult offspring reproduction. We show that genes that regulate serotonin signaling and action in the ovary are altered in prenatally FLX-exposed offspring, which when coupled with increased expression of components of the core Circadian Locomotor Output Cycles Kaput (CLOCK) gene regulatory loop may suggest an interaction between serotonergic signaling and clock gene signaling pathways leading to the altered reproductive phenotype.

Background The key to understanding changes in gene expression levels using reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR) relies on the ability to rationalize the technique using internal control genes (ICGs). However, the use of ICGs has become increasingly problematic given that any genes, including housekeeping genes, thought to be stable across different tissue types, ages and treatment protocols, can be regulated at transcriptomic level. Our interest in prenatal glucocorticoid (GC) effects on fetal growth has resulted in our investigation of suitable ICGs relevant in this model. The usefulness of RNA18S, ACTB, HPRT1, RPLP0, PPIA and TUBB as ICGs was analyzed according to effects of early dexamethasone (DEX) treatment, gender, and gestational age by two approaches: (1) the classical approach where raw (i.e., not normalized) RT-qPCR data of tested ICGs were statistically analyzed and the best ICG selected based on absence of any significant effect; (2) used of published algorithms. For the latter the geNorm Visual Basic application was mainly used, but data were also analyzed by Normfinder and Bestkeeper. In order to account for confounding effects on the geNorm analysis due to co-regulation among ICGs tested, network analysis was performed using Ingenuity Pathway Analysis software. The expression of RNA18S, the most abundant transcript, and correlation of ICGs with RNA18S, total RNA, and liver-specific genes were also performed to assess potential dilution effect of raw RT-qPCR data. The effect of the two approaches used to select the best ICG(s) was compared by normalization of NR3C1 (glucocorticoid receptor) mRNA expression, as an example for a target gene. Results Raw RT-qPCR data of all the tested ICGs was significantly reduced across gestation. TUBB was the only ICG that was affected by DEX treatment. Using approach (1) all tested ICGs would have been rejected because they would initially appear as not reliable for normalization. However, geNorm analysis (approach 2) of the ICGs indicated that the geometrical mean of PPIA, HPRT1, RNA18S and RPLPO can be considered a reliable approach for normalization of target genes in both control and DEX treated groups. Different subset of ICGs were tested for normalization of NR3C1 expression and, despite the overall pattern of the mean was not extremely different, the statistical analysis uncovered a significant influence of the use of different normalization approaches on the expression of the target gene. We observed a decrease of total RNA through gestation, a lower decrease in raw RT-qPCR data of the two rRNA measured compared to ICGs, and a positive correlation between raw RT-qPCR data of ICGs and total RNA. Based on the same amount of total RNA to performed RT-qPCR analysis, those data indicated that other mRNA might have had a large increase in expression and, as consequence, had artificially diluted the stably expressed genes, such as ICGs. This point was demonstrated by a significant negative correlation of raw RT-qPCR data between ICGs and liver-specific genes. Conclusion The study confirmed the necessity of assessing multiple ICGs using algorithms in order to obtain a reliable normalization of RT-qPCR data. Our data indicated that the use of the geometrical mean of PPIA, HPRT1, RNA18S and RPLPO can provide a reliable normalization for the proposed study. Furthermore, the dilution effect observed support the unreliability of the classical approach to test ICGs. Finally, the observed change in the composition of RNA species through time reveals the limitation of the use of ICGs to normalize RT-qPCR data, especially if absolute quantification is required. Electronic supplementary material The online version of this article (doi:10.1186/s13104-015-0973-7) contains supplementary material, which is available to authorized users.

Glucocorticoid treatment given in late pregnancy in sheep resulted in altered placental development and function. An imbalance of placental survival and apoptotic factors resulting in an increased rate of apoptosis may be involved. We have now investigated the effects of dexamethasone (DEX) in early pregnancy on binucleate cells (BNCs), placental apoptosis, and fetal sex as a determinant of these responses. Pregnant ewes carrying singleton fetuses (n = 105) were randomized to control (n = 56, 2 mL saline/ewe) or DEX treatment (n = 49, intramuscular injections of 0.14 mg/kg ewe weight per 12 hours over 48 hours) at 40 to 41 days of gestation (dG). Placentomes were collected at 50, 100, 125, and 140 dG. At 100 dG, DEX in females reduced BNC numbers, placental antiapoptotic (proliferating cell nuclear antigen), and increased proapoptotic factors (Bax, p53), associated with a temporarily decrease in fetal growth. At 125 dG, BNC numbers and apoptotic markers were restored to normal. In males, ovine placental lactogen-protein levels after DEX were increased at 50 dG, but at 100 and 140 dG significantly decreased compared to controls. In contrast to females, these changes were independent of altered BNC numbers or apoptotic markers. Early DEX was associated with sex-specific, transient alterations in BNC numbers, which may contribute to changes in placental and fetal development. Furthermore, in females, altered placental apoptosis markers may be involved.

Fetal exposure to synthetic glucocorticoids in sheep results in increased fetal hepatic 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) and corticosteroid-binding globulin (CBG) protein levels and insulin resistance in postnatal life. The aim was to determine whether these changes persisted to adulthood and whether alterations in mediators of hepatic glucocorticoid and glucose regulation contributed to changes in metabolism. Pregnant ewes or their fetuses received either repeated intramuscular saline (MS, FS) or betamethasone injections (0.5 mg/kg; M4, F4) at 104, 111, 118, and 124 days of gestation (dG), or a single betamethasone injection at 104 dG followed by saline at 111, 118, and 124 dG (M1, F1). Offspring were catheterized at 2 and 3 yr of age and given an intravenous glucose challenge (0.5 mg/kg). Hepatic tissue was collected at 3.5 yr. At 2 yr of age, basal plasma insulin was elevated in M4 offspring and at 3 yr of age was elevated in F4 offspring. Basal insulin-to-glucose ratio was significantly elevated in M4 offspring at 2 yr of age and elevated in M1, M4, and F4 offspring at 3 yr of age. All betamethasone treatments resulted in significant increases in hepatic glucose-6-phosphatase (G-6-Pase) activity. Hepatic glucocorticoid receptor protein levels were not altered in M1 and M4 offspring but were increased in F1 and F4 offspring. Hepatic CBG protein levels were lower in F4 but not F1 offspring and were unchanged from control in M1 and M4 offspring. Prenatal betamethasone exposure results in elevated hepatic G-6-Pase activity in adulthood and may contribute to long-term changes in metabolism.

There is now considerable epidemiological and experimental evidence indicating that early-life environmental conditions, including nutrition, affect subsequent development in later life. These conditions induce highly integrated responses in endocrine-related homeostasis, resulting in persistent changes in the developmental trajectory producing an altered adult phenotype. Early-life events trigger processes that prepare the individual for particular circumstances that are anticipated in the postnatal environment. However, where the intrauterine and postnatal environments differ markedly, such modifications to the developmental trajectory may prove maladaptive in later life. Reproductive maturation and function are similarly influenced by early-life events. This should not be surprising, because the primordial follicle pool is established early in life and is thus vulnerable to early-life events. Results of clinical and experimental studies have indicated that early-life adversity is associated with a decline in ovarian follicular reserve, changes in ovulation rates, and altered age at onset of puberty. However, the underlying mechanisms regulating the relationship between the early-life developmental environment and postnatal reproductive development and function are unclear. This review examines the evidence linking early-life nutrition and effects on the female reproductive system, bringing together clinical observations in humans and experimental data from targeted animal models.

The long-term outcomes of antenatal glucocorticoids (GCs) vary between reports, and have generated controversy in terms of repeated and single-course events, causing irreversible effects on endocrine set points.This study aimed to assess the effects of alternative therapeutic agents other than synthetic glucocorticoid GC administration for fetal lung maturation.A review of literature from PubMed, EMBASE, Cochrane Library, and Google Scholar was conducted to assess the use of alternative therapies to synthetic GCs using recommendations of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses statement (PRISMA). End points included the rates of respiratory distress syndrome (RDS), mRNA expression for pneumocyte type II, concentration of surfactant proteins in alveolar lavage, morphological differences, histological proof of lung maturation, and angiogenesis or quantification of the surfactant pool.In all 41 studies examined, we found that ambroxol showed positive effects on lung maturation, but it has yet to be analyzed with sufficient significance in humans. Interleukins and TNF-alpha produce accelerated lung maturation, but have only been evaluated in basic research/experimental studies. Growth factors promote structural and functional growth in all phases of lung maturation, but little is known about their reciprocal effects and exact mechanisms as therapeutics. Thyroid releasing hormone or vitamin A cause detrimental side effects or were less effective for lung maturation.The efficacy and safety of these alternative agents are differentiated and none up to now can be recommended as an alternative to GCs.

Maternal nutritional restriction has been shown to induce impairments in a number of organ systems including the ovary. We have previously shown that maternal undernutrition induces fetal growth restriction and low birth weight, and results in an offspring ovarian phenotype characteristic of premature ovarian aging with reduced ovarian reserve. In the present study, we set out to investigate the underlying mechanisms that lead offspring of undernourished mothers to premature ovarian aging. Pregnant dams were randomized to 1) a standard diet throughout pregnancy and lactation (control), 2) a calorie-restricted (50% of control) diet during pregnancy, 3) a calorie-restricted (50% of control) diet during pregnancy and lactation, or 4) a calorie-restricted (50% of control) diet during lactation alone. The present study shows that early life undernutrition-induced reduction of adult ovarian follicles may be mediated by increased ovarian endoplasmic reticulum stress in a manner that increased follicular apoptosis but not autophagy. These changes were associated with a loss of ovarian vessel density and are consistent with an accelerated ovarian aging phenotype. Whether these changes are mediated specifically by a reduction in the local antioxidant environment is unclear, although our data suggest the possibility that ovarian melatonin may play a part in early life nutritional undernutrition and impaired offspring folliculogenesis.

In this study, we determined the gene and/or protein expression of hypothalamic–pituitary–adrenal (HPA) axis regulatory molecules following synthetic glucocorticoid exposures. Pregnant sheep received intramuscular saline or betamethasone (BET) injections at 104 (BET-1), 104 and 111(BET-2) or 104, 111 and 118 (BET-3) days of gestation (dG). Samples were collected at numerous time-points between 75 dG and 12 weeks postnatal age. In the BET-3 treatment group, fetal plasma cortisol levels were lower at 145 dG than controls and gestational length was lengthened significantly. The cortisol:adrenocorticotropic hormone (ACTH) ratio in fetal plasma of control and BET-3 fetuses rose significantly between132 and 145 dG, and remained elevated in lambs at 6 and 12 weeks of age; this rise was truncated at day 145 in fetuses of BET-3 treated mothers. After BET treatment, fetal and postnatal pituitary proopiomelanocortin mRNA levels were reduced from 109 dG to 12 weeks postnatal age; pituitary prohormone convertase 1 and 2 mRNA levels were reduced at 145 dG and postnatally; hypothalamic arginine vasopressin mRNA levels were lowered at all time-points, but corticotrophin-releasing hormone mRNA levels were reduced only in postnatal lambs. Maternal BET increased late fetal and/or postnatal adrenal mRNA levels of ACTH receptor and 3β hydroxysteroid dehydrogenase but decreased steroidogenic acute regulatory protein and P450 17-α hydroxylase. The altered mRNA levels of key HPA axis regulatory proteins after maternal BET injections suggests processes that may subserve long-term changes in HPA activity in later life after prenatal exposure to synthetic glucocorticoids.

Excessive fructose consumption is associated with insulin resistance (IR) and nonalcoholic fatty liver disease (NAFLD), and high fructose intake during pregnancy can lead to compromised fetal development in the rat. Evidence suggests that the amino acid taurine can ameliorate fructose-induced IR and NAFLD in nonpregnant animals. This study investigated the efficacy of taurine supplementation on maternal fructose-induced metabolic dysfunction and neonatal health. Time-mated Wistar rats were randomized to four groups during pregnancy and lactation: (a) control diet (CON), (b) CON supplemented with 1.5% taurine in drinking water (CT), (c) CON supplemented with fructose solution (F) and (d) F supplemented with taurine (FT). Maternal and neonatal weights, plasma cytokines and hepatic gene expression were analyzed. Maternal hyperinsulinemia, increased homeostasis model assessment of IR indices and elevated proinflammatory cytokines were observed in F group and normalized in FT group. Maternal fructose-induced hepatic steatosis accompanied with increased liver weight was ameliorated with taurine supplementation. Maternal hepatic sterol regulatory element-binding protein-1c and fatty acid synthase expression was significantly increased in the F group compared to the CON, CT and FT groups. Neonatal hepatic phosphoenolpyruvate carboxykinase expression was increased in male F neonates compared to the CON, CT and FT groups and was increased in female F and FT neonates compared to CON and CT. Interleukin-1β expression was decreased in male CT and FT neonates compared to other male groups. Hepatic tumour necrosis factor receptor-1 was lower in the male FT group than the F group. These results demonstrate that maternal taurine supplementation can partially reverse fructose-induced maternal metabolic dysfunction and may ameliorate adverse developmental programming effects in offspring in a sex-specific manner.

The consumption of artificially sweetened processed foods, particularly high in fructose or high fructose corn syrup, has increased significantly in the past few decades. As such, interest into the long term outcomes of consuming high levels of fructose has increased significantly, particularly when the exposure is early in life. Epidemiological and experimental evidence has linked fructose consumption to the metabolic syndrome and associated comorbidities—implicating fructose as a potential factor in the obesity epidemic. Yet, despite the widespread consumption of fructose-containing foods and beverages and the rising incidence of maternal obesity, little attention has been paid to the possible adverse effects of maternal fructose consumption on the developing fetus and long term effects on offspring. In this paper we review studies investigating the effects of fructose intake on metabolic outcomes in both mother and offspring using human and experimental studies.

International audience; A general characteristic of fetal endocrine maturation across different species is the enhanced activity of the fetal hypothalamic-pituitary-adrenal (HPA) axis during late gestation. Precocious activation of this axis may occur when the fetus is exposed to an adverse intra-uterine environment, such as hypoxemia. HPA development is associated with increased levels of ACTH(1-39) and adrenal corticosteroids (cortisol in sheep and human) in the fetal circulation, and increased expression of mRNA encoding corticotrophin releasing hormone (CRH) in the hypothalamus, proopiomelanocortin (POMC) in the pituitary, and key steroidogenic enzymes in the fetal adrenal. At term, increased levels of cortisol act on the placenta/trophoblast derived cells to increase expression of prostaglandin synthase Type II (PGHS-II). In human gestation, cortisol also decreases expression of 15-hydroxyprostaglandin dehydrogenase (PGDH) in chorionic trophoblast cells. Increased synthesis and decreased metabolism of prostaglandin (PG) results, during late gestation, in enhanced output of primary PG, which in turn increases the activity of 11 beta-hydroxysteroid dehydrogenase (11 beta HSD) in the human fetal membranes. Increased chorionic 11 beta HSD-1 results in increased local generation of cortisol from cortisone, with further paracrine/autocrine stimulation of PG output. Increased fetal cortisol contributes to the maturation of organ systems required for postnatal extra-uterine survival. However, excessive levels of feto-placental glucocorticoid, derived from maternal administration of synthetic corticosteroids or sustained endogenous fetal cortisol production, results in intrauterine growth restriction. Fetal sheep, exposed to maternal betamethasone in late gestation, develop insulin resistance and exaggerated adrenal responses to HPA stimulation by 6-12 months postnatal life. Thus, the level of fetal HPA activity is crucial not only for determining gestation length, but may also predict pathophysiologic adjustments in later life.

Synthetic glucocorticoids have become an important clinical tool with which to advance fetal lung maturation in women at risk of early preterm birth, and this has succeeded in reducing neonatal mortality and morbidity from respiratory distress syndrome. Although previous studies have shown that glucocorticoids have deleterious consequences on fetal development, there is little information regarding the effects of clinically relevant repeated maternal doses of glucocorticoids on fetal growth and hypothalamic-pituitary-adrenal (HPA) function. We hypothesised that repeated prenatal exposure to increased concentrations of glucocorticoids would alter fetal growth and HPA axis development. Pregnant ewes were injected with betamethasone (0.5 mg/kg) or vehicle at 104, 111 and 118 days of gestation (term 150 days). Animals were sacrificed at 125 and 146 days of gestation, at which time fetal weights were recorded. Maternal and fetal blood samples were gathered and fetal tissue collected. Maternal oestradiol concentrations were significantly greater than those in controls at 125 days of gestation, but were not different at 146 days. Maternal plasma progesterone concentrations were similar between groups at both 125 and 146 days of gestation. Weight at birth was significantly reduced by 23% at 125 days and 19% at 146 days of gestation (P

International audience; Birth in most animal species is triggered by the fetus through activation of the fetal hypothalamic-pituitary-adrenal (HPA) axis. Preterm birth, may be associated with precocious activation of fetal HPA function, reflecting the fetal response to an adverse intrauterine environment. There is a progressive and concurrent increase of ACTH1-39 and cortisol (F) in the circulation of fetal sheep during the last 15-20 days of pregnancy (term, day 145-150) associated with increased expression of hypothalamic CRH pituitary POMC and adrenal ACTH receptor and steroidogenic enzymes, particularly P450 C17. Similar changes occur with fetal hypoxemia. Negative feedback is ameliorated by decreased pituitary and hypothalamic glucocorticoid receptor, increased CBG, and altered fetal pituitary 11B-hydroxysteroid dehydrogenase type 1. Repeated fetal hypoxemia, diminishes the fetal-pituitary ACTH response, but increases fetal adrenal responsiveness. Fetuses exposed to maternal glucocorticoid in late gestation are growth restricted with altered postnatal HPA responsiveness and glycemic responses that reproduce the insulin resistance of type 2 diabetes. We conclude that the level of fetal HPA activity is crucial not only for determining gestation length, but also predicts pathophysiologic adjustment in later life.

An adverse early-life environment is associated with long-term disease consequences. Adversity early in life is hypothesized to elicit developmental adaptations that serve to improve fetal and postnatal survival and prepare the organism for a particular range of postnatal environments. These processes, although adaptive in their nature, may later prove to be maladaptive or disadvantageous if the prenatal and postnatal environments are widely discrepant. The exposure of the fetus to elevated levels of either endogenous or synthetic glucocorticoids is one model of early-life adversity that contributes substantially to the propensity of developing disease. Moreover, early-life glucocorticoid exposure has direct clinical relevance because synthetic glucocorticoids are routinely used in the management of women at risk of early preterm birth. In this regard, reports of adverse events in human newborns have raised concerns about the safety of glucocorticoid treatment; synthetic glucocorticoids have detrimental effects on fetal growth and development, childhood cognition, and long-term behavioral outcomes. Experimental evidence supports a link between prenatal exposure to synthetic glucocorticoids and alterations in fetal development and changes in placental function, and many of these alterations appear to be permanent. Because the placenta is the conduit between the maternal and fetal environments, it is likely that placental function plays a key role in mediating effects of fetal glucocorticoid exposure on hypothalamic-pituitary-adrenal axis development and long-term disease risk. Here we review recent insights into how the placenta responds to changes in the intrauterine glucocorticoid environment and discuss possible mechanisms by which the placenta mediates fetal hypothalamic-pituitary-adrenal development, metabolism, cardiovascular function, and reproduction.

Maternal obesity is associated with obesity and metabolic disorders in offspring. However, intervention strategies to reverse or ameliorate the effects of maternal obesity on offspring health are limited. Following maternal undernutrition, taurine supplementation can improve outcomes in offspring, possibly via effects on glucose homeostasis and insulin secretion. The effects of taurine in mediating inflammatory processes as a protective mechanism has not been investigated. Further, the efficacy of taurine supplementation in the setting of maternal obesity is not known. Using a model of maternal obesity, we examined the effects of maternal taurine supplementation on outcomes related to inflammation and lipid metabolism in mothers and neonates. Time-mated Wistar rats were randomised to either: 1) control : control diet during pregnancy and lactation (CON); 2) CON supplemented with 1.5% taurine in drinking water (CT); 3) maternal obesogenic diet (high fat, high fructose) during pregnancy and lactation (MO); or 4) MO supplemented with taurine (MOT). Maternal and neonatal weights, plasma cytokines and hepatic gene expression were analysed. A MO diet resulted in maternal hyperinsulinemia and hyperleptinemia and increased plasma glucose, glutamate and TNF-α concentrations. Taurine normalised maternal plasma TNF-α and glutamate concentrations in MOT animals. Both MO and MOT mothers displayed evidence of fatty liver accompanied by alterations in key markers of hepatic lipid metabolism. MO neonates displayed a pro-inflammatory hepatic profile which was partially rescued in MOT offspring. Conversely, a pro-inflammatory phenotype was observed in MOT mothers suggesting a possible maternal trade-off to protect the neonate. Despite protective effects of taurine in MOT offspring, neonatal mortality was increased in CT neonates, indicating possible adverse effects of taurine in the setting of normal pregnancy. These data suggest that maternal taurine supplementation may ameliorate the adverse effects observed in offspring following a maternal obesogenic diet but these effects are dependent upon prior maternal nutritional background.

Alterations in early life nutrition lead to an increased risk of obesity and metabolic syndrome in offspring. We have shown that both relative maternal undernutrition (UN) and maternal obesity result in metabolic derangements in offspring, independent of the postnatal dietary environment. Since insulin-like growth factor binding protein 2 (IGFBP2) has been shown to be independently associated with obesity and diabetes risk, we examined the IGF–IGFBP axis in male rat offspring following either maternal UN or maternal obesity to explain possible common pathways in the development of metabolic disorders. Wistar rats were time-mated and fed either a control diet (CONT), 50 % of CONT (UN) or a high-fat (HF) diet throughout pregnancy. Male offspring were weaned onto a standard chow diet and blood and tissues were collected at postnatal day 160. Plasma and hepatic tissue samples were analysed for key players in the IGF–IGFBP system. Both maternal UN and HF resulted in increased fat mass, hyperinsulinemia, hyperleptinemia and altered blood lipid profiles in offspring compared to CONT. Circulating IGF-1 and IGFBP3 levels and hepatic mRNA expression of IGFBP1 and IGFBP2 were significantly decreased in UN and HF offspring compared to CONT. DNA methylation of the IGFBP2 promotor region was similar between maternal dietary groups. Although chaperone gene heat-shock protein 90 and hepatic IGFBP1 were significantly correlated in CONT offspring this effect was absent in both UN and HF offspring. In conclusion, this study is one of the first to directly compare two experimental models of developmental programming representing both ends of the maternal dietary spectrum. Our data suggest that two disparate nutritional models that elicit similar adverse metabolic phenotypes in offspring are characterised by common alterations in the IGF–IGFBP pathway.

Contents Pre-natal glucocorticoids are used in women at risk of preterm delivery to induce foetal lung maturation. However, glucocorticoids can produce negative outcomes for other tissues such as the reproductive system. We therefore tested the effects of pre-natal betamethasone on testicular morphology and apoptotic protein immune expression during pre- and post-natal development. Pregnant ewes (n = 42) bearing singleton male foetuses were randomly allocated to receive intramuscular injections of saline or betamethasone (0. 5 mg/kg) at 104, 111 and 118 days of gestation (DG). Testes were collected at 121 and 132 DG, and at 45 and 90 post-natal days (PD) and subjected to morphometric analysis (volume densities of sex cords and interstitial tissues; sex cord diameter). Immunohistochemistry (% stained area) was used to assess active caspase-3, Bax, Bcl-2 and cell-cycle proteins (PCNA). Compared with control values, betamethasone treatment decreased sex cord diameter at 121 DG, 45 and 90 PD, and sex cord volume at 90 PD. Active caspase-3 was decreased by betamethasone at 121 DG and 90 PD, but Bax was increased in all betamethasone groups. Bcl-2 and PCNA decreased in the betamethasone groups at 121 DG and 45 PD, but increased at 132 DG and 90 PD. We conclude that high levels of pre-natally administered glucocorticoid reduce foetal testicular development, perhaps via changes in the balance between pro- and anti-apoptotic proteins and cell-cycle proteins. These outcomes could compromise the future spermatogenic potential of male offspring.

Considerable epidemiological, experimental and clinical data have amassed showing that the risk of developing disease in later life is dependent upon early life conditions. In particular, altered maternal nutrition, including undernutrition and overnutrition, can lead to metabolic disorders in offspring characterised by obesity and leptin resistance. The adipokine leptin has received significant interest as a potential programming factor; alterations in the profile of leptin in early life are associated with altered susceptibility to obesity and metabolic disorders in adulthood. Maintenance of a critical leptin level during early development facilitates the normal maturation of tissues and signalling pathways involved in metabolic homeostasis. A period of relative hypo- or hyperleptinemia during this window of development will induce some of the metabolic adaptations which underlie developmental programming. However, it remains unclear whether leptin alone is a critical factor for the programming of obesity. At least in animal experimental studies, developmental programming is potentially reversible by manipulating the concentration of circulating leptin during a critical window of developmental plasticity and offers an exciting new approach for therapeutic intervention.

The effects of maternal fructose intake on offspring health remain largely unknown, despite the marked increase in consumption of sweetened beverages that has paralleled the obesity epidemic. The present study investigated the impact of maternal fructose intake on placental, fetal, and neonatal development. Female Wistar rats were time-mated and allocated to receive either water [control (CONT)] or fructose solution designed to provide 20% of caloric intake from fructose (FR). FR was administered from d 1 of pregnancy until postnatal day (P) 10. All dams had ad libitum access to standard laboratory chow and water. Dams and offspring were killed at embryonic day (E) 21 and P10. FR dams demonstrated increased total caloric intake and maternal hyperinsulinemia at E21 as well as increased maternal plasma fructose levels at E21 and P10. FR intake did not alter maternal blood glucose, β-hydroxybutyrate (BHB), or electrolyte levels at either time point. Fetal weights at E21 were unchanged, although placental weights were reduced in FR female but not FR male fetuses. Plasma leptin, fructose, and blood glucose levels were increased and BHB levels decreased in FR female but not male fetuses. Plasma insulin levels were not different between CONT and FR groups. Male and female FR neonates had higher plasma fructose levels and were hypoinsulinemic but euglycemic at P10 compared with CONT. Blood BHB levels were increased in FR male neonates but not females at P10. P10 plasma leptin levels were not different between groups. Stomach content leptin levels were increased in all FR offspring at P10, but no differences in stomach content insulin or fructose levels were observed. This study reports for the first time that maternal FR intake resulted in sex-specific changes in offspring development, whereby females appear more vulnerable to metabolic compromise during neonatal life. Independent follow-up studies are essential to investigate the long-term consequences of maternal FR consumption on offspring health.