Wild-type Dams Research Articles

Leprdb/+ Dams Protect Wild-type Male Offspring Bone Strength from the Detrimental Effects of a High-Fat Diet.

The prevalence of maternal obesity is increasing at an alarming rate and increases the life-long risk of developing cardiometabolic disease in adult offspring. Leptin, an adipokine, is systemically elevated in the obese milieu. We recently showed that maternal hyperleptinemia without obesity improves offspring insulin sensitivity and glucose tolerance while protecting against weight gain on a high-fat, high-sugar (HFD). Here, we investigate the effect of maternal hyperleptinemia on offspring bone by using 2 independent maternal models. First, we compared wild-type (WT) offspring from severely hyperleptinemic Leprdb/+ (DB/+) dams with those from WT dams. In the second model, WT females were implanted with miniosmotic pumps that released either saline (group SAL) or leptin (group LEP; 650ng/hour) and the WT offspring were compared. At 23 weeks of age, a subset of offspring were challenged with a HFD for 8 weeks. When the offspring were 31 weeks of age, bone geometry, strength, and material properties were investigated. The HFD increased trabecular bone volume but decreased both total breaking strength and material strength of femora from the offspring of WT dams. However, male offspring of DB/+ dams were protected from the detrimental effects of a HFD, while offspring of LEP dams were not. Further material analysis revealed a modest decrease in advanced glycation end product accumulation coupled with increased collagen crosslinking in male offspring from DB/+ dams on a HFD. These data suggest that while maternal leptin may protect bone quality from the effects of a HFD, additional factors of the maternal environment controlled by leptin receptor signaling are likely also involved.

Milk Osteopontin Promotes Intestinal Development in Early Life by Increasing Expression of Osteopontin Receptors

Abstract Objectives Osteopontin (OPN), a phosphorylated glycoprotein, is abundantly present in both human and mouse milk, making the mouse a good model to study functions of milk OPN. OPN is a pleiotropic protein involved in many biological processes such as cell proliferation and differentiation. OPN initiates signaling pathways to exert its multiple functions by binding to its receptors on the surface of target cells. Since milk OPN is partly resistant to in vitro digestion, the current study aimed to investigate roles of milk OPN on intestinal development in early life. Methods A mouse model established by our group was used. In this model, wild-type (WT) mouse pups were either nursed by WT or OPN knock-out (KO) dams, receiving milk with or without OPN from postnatal days 0 to 21 (P0−P21). Whether milk OPN is resistant to in vivo digestion and effects of milk OPN on intestinal development were examined. Results Milk OPN was resistant to in vivo digestion as indicated by immunoblotting using intestinal contents from P12 pups. Young pups (P4 and P6) nursed by WT dams (OPN + group) exhibited significantly longer small intestines than did pups nursed by OPN KO dams (OPN- group), but older pups had similar lengths in both groups (P8 to P30). Further, OPN + pups had a larger inner surface than did OPN- pups at P10 and P20 as revealed by histological analysis of the ratio of villus height/crypt depth. The activity of an intestinal differentiation marker (alkaline phosphatase) was significantly higher in the OPN + group than the OPN- group at P30. In addition, compared with the OPN- group, the OPN + pups (P30) had better differentiation based on the observation that more goblet cells and Paneth cells appeared in the small intestine.. To determine how milk OPN exerts its functions on the intestine, effects of milk OPN on expression of OPN receptors were then determined. qRT-PCR results showed that milk OPN increased transcription of OPN receptors, including integrin αv, integrin β3, and CD44 in duodenum and jejunum from P4 to P30 mouse pups. Immunoblotting results also indicated that milk OPN increased the protein levels of integrin αv. Conclusions Milk OPN promotes intestinal proliferation and differentiation by up-regulating expression of OPN receptors in early life. Funding Sources This study was in part funded by Mead Johnson Nutrition.

Glutathione Peroxidase Influence on Cardiac Remodeling in Progeny of Dams Exposed to Engineered Nanomaterials during Gestation

Vulnerable populations, like the developing fetus, may be more susceptible to sustained detriments elicited by inhaled engineered nanomaterials (ENMs) entering the bloodstream and prompting systemic effects. Previous studies have demonstrated diminished mitochondrial bioenergetics in progeny following maternal exposure to a highly utilized ENM, nano‐titanium dioxide (nano‐TiO2). Cardiac dysfunction and reduced ROS scavenging ability were observed with decreased mitochondria phospholipid hydroperoxide glutathione peroxidase (mPHGPx) expression. This study aimed to elucidate whether overexpression of mPHGPx could provide sufficient antioxidant protection to attenuate cardiac dysfunction in pregnant dams and progeny following gestational ENM inhalation exposure. Wild‐type (WT) and transgenic (mPHGPx) female mice were bred with transgenic and WT males, respectively, to delineate maternal vs progeny protection. Pregnant dams were exposed to nano‐TiO2 (6 hrs/day for 6 days) with a primary particle size of 21nm and mass concentration of 12mg/m3 or sham air. Conventional (M‐Mode) and speckle tracking stress‐strain echocardiographic parameters were assessed in pregnant dams, as well as in fetal (gestational day 14) and adult (11 wks) offspring. Following inhalation exposure to nano‐TiO2 during gestation, WT dams exhibited signs of concentric remodeling marked by significantly decreased (left ventricular) LV diastolic diameter (12%) and volume (26%), and significant increases in both LV posterior wall thickness (23%) and relative wall thickness (39%), which were preserved in mPHGPx pregnant dams. Fetal offspring of mPHGPx dams had significantly enhanced cardiac output (87%) and stroke volume (43%) compared to fetal offspring of WT dams. Stress‐strain analysis revealed that fetal WT offspring of nano‐TiO2–exposed mPHGPx dams had significantly higher short‐axis systolic circumferential strain rate (~3 fold) and displacement (~2 fold) than WT offspring of WT dams. Adult WT offspring of transgenic dams presented with a significantly lower ejection fraction (6% and 8%) and fractional shortening (18% and 22%) than sham WT and nano‐TiO2 transgenic offspring, respectively. This may suggest that in offspring lacking the transgene, enhanced maternal antioxidant defense alone may not provide sufficient protection into adulthood. This study highlights the beneficial role of enhanced antioxidant defense (during fetal development and adulthood) on cardiac dysfunction following gestational ENM exposure.Support or Funding InformationSupported by: R01 HL‐128485 (JMH), CFO for the Ohio Valley Whipkey Trust (JMH), R01‐ES015022 (TRN), AHA‐17PRE33660333 (QAH).

Maternal Supplementation of Inositols, Fucoxanthin, and Hydroxytyrosol in Pregnant Murine Models of Hypertension.

Myoinositol (M) and D-chiro-inositol (D) are insulin sensitizer compounds, while fucoxanthin (F) and hydroxytyrosol (H) are antioxidant substances. We aim to investigate if the combination of these compounds, will improve the vascular responses in pregnant mouse models of hypertension: a genetic model, transgenic heterozygous mice lacking endothelial nitric oxide synthase gene (eNOS-/+); and environmental, wild-type (WT) mice. Those mouse models will allow a better understanding of the genetic/environmental contribution to hypertension in pregnancy. eNOS-/+ and WT female were fed high fat diet for 4 weeks, then at 7-8 weeks of age were mated with WT male. On gestational day (GD) 1, they were randomly allocated to receive MDFH treatment or water as control: eNOS-/+ MDFH (n = 13), eNOS-/+ (n = 13), WT-MDFH (n = 14), and WT (n = 20). Systolic blood pressure (SBP) was obtained at GD 18, then dams were sacrificed; fetuses and placentas collected, and 2 mm segments of carotid arteries isolated for vascular responses using the wire-myograph system. Responses to phenylephrine (PE), with/without the NOS inhibitor (N-nitro-l-arginine methyl ester (l-NAME)), and to acetylcholine (Ach) and sodium nitroprussiate (SNP) were performed. SBP decreased in eNOS-/+ and WT dams after MDFH supplementation. In eNOS-/+, MDFH lower the contractile response to PE and l-NAME and improved Ach vasorelaxation. In WT dams, MDFH treatment did not affect PE response; MDFH treatment lowered the vascular PE response after incubation with l-NAME. No differences were seen in SNP relaxation in both models. MDFH decreased SBP in both genetically and environmentally hypertensive dams and improved vascular responses mostly in the eNOS-/+ dams.

Maternal Fat-1 Transgene Protects Offspring from Excess Weight Gain, Oxidative Stress, and Reduced Fatty Acid Oxidation in Response to High-Fat Diet.

Overweight and obesity accompanies up to 70% of pregnancies and is a strong risk factor for offspring metabolic disease. Maternal obesity-associated inflammation and lipid profile are hypothesized as important contributors to excess offspring liver and skeletal muscle lipid deposition and oxidative stress. Here, we tested whether dams expressing the fat-1 transgene, which endogenously converts omega-6 (n-6) to omega-3 (n-3) polyunsaturated fatty acid, could protect wild-type (WT) offspring against high-fat diet induced weight gain, oxidative stress, and disrupted mitochondrial fatty acid oxidation. Despite similar body mass at weaning, offspring from fat-1 high-fat-fed dams gained less weight compared with offspring from WT high-fat-fed dams. In particular, WT males from fat-1 high-fat-fed dams were protected from post-weaning high-fat diet induced weight gain, reduced fatty acid oxidation, or excess oxidative stress compared with offspring of WT high-fat-fed dams. Adult offspring of WT high-fat-fed dams exhibited greater skeletal muscle triglycerides and reduced skeletal muscle antioxidant defense and redox balance compared with offspring of WT dams on control diet. Fat-1 offspring were protected from the reduced fatty acid oxidation and excess oxidative stress observed in offspring of WT high-fat-fed dams. These results indicate that a maternal fat-1 transgene has protective effects against offspring liver and skeletal muscle lipotoxicity resulting from a maternal high-fat diet, particularly in males. Altering maternal fatty acid composition, without changing maternal dietary composition or weight gain with high-fat feeding, may highlight important strategies for n-3-based prevention of developmental programming of obesity and its complications.

Evaluation of Bioactivities of Bovine Milk Osteopontin Using a Knockout Mouse Model.

Osteopontin (OPN) is a multifunctional protein present abundantly in human milk, but at low levels in bovine milk and infant formula. Bovine milk OPN (bmOPN) is commercially available, and may therefore, be added to formula. OPN exerts its multiple functions by binding to its receptors to activate cell signaling pathways. The OPN receptor (integrin)-binding site is conserved across species; therefore, bmOPN may exert bioactivities in humans and mice. The objective of the present study was to evaluate bioactivities of bmOPN using an established OPN knock-out (KO) mouse model. We evaluated bioactivities of bmOPN, including effects on intestinal growth, immune response, and brain development. In the present study, wild-type (WT) pups were nursed by WT dams, KO dams, or KO dams with bmOPN supplementation from postnatal days 1 to 21 (P1--P21). Our results show that orally ingested bmOPN is partly resistant to in vivo gastrointestinal digestion, and supplemental bmOPN exhibited similar effects as mouse milk OPN (mmOPN) on promoting growth of the small intestine revealed by histological analysis of duodenum villus height and crypt depth at P10, on modifying TNF-α response against a LPS challenge at P30, as well as promoting brain myelination by increasing expression of myelin-associated glycoprotein (MAG) and myelin basic protein (MBP) and improving cognitive development. Our finding that bmOPN with an amino acid sequence different from mmOPN but with a conserved integrin binding site exerts bioactivities similar to mmOPN suggests that bmOPN may provide bioactivities to human infants when added to formula.

Toll-like receptor 2 impacts the development of oral tolerance in mouse pups via a milk-dependent mechanism

The role of breast-feeding in the development of oral tolerance and allergic diseases is controversial, which could be related to variability in milk components. Toll-like receptor 2 (TLR2) is an innate immune receptor implicated in regulating allergic disease development. We examined whether deficiency of maternal TLR2 affects the normal development of oral tolerance and related immune parameters during lactation in a mouse model. Heterozygous TLR2+/- pups from wild-type (WT) or TLR2-/- dams were fed either by their biologic dam or a dam of the alternate genotype. Development of oral tolerance to ovalbumin, levels of tolerogenic CD103+ dendritic cells, and regulatory T (Treg) cells, as well as intestinal permeability, were evaluated in these pups. The levels of key immune mediators in milk from TLR2-/- and WT mothers were also examined. Heterozygous TLR2+/- pups that were born to and nursed by TLR2-/- dams exhibited impaired oral tolerance. This was prevented by cross-fostering onto WT (TLR2+/+) dams. Impairments included selective elevation in anti-ovalbumin IgE in plasma following immunization, reduced numbers of tolerogenic dendritic cells and Treg cells in the intestinal tract, and increased intestinal permeability. TLR2 deficiency also affected milk content of insulin-like growth factor-1, IFN-γ, IL-6, and IL-13. Our results underline a critical role for TLR2 in regulating milk components that are essential for development of oral tolerance in early life and demonstrate the importance of considering the immune status of nursing mothers in studies of immune development and responses.

Transcriptomic analysis investigating the interaction between peripheral serotonin and high-fat diet feeding on mammary gene expression in midlactation mice.

To understand the role of peripheral serotonin and its interaction with diet in midlactation mammary gene expression, our study uses tryptophan hydroxylase 1 knockout (Tph1-KO) mice fed a high-fat diet (HFD). It has previously been demonstrated that HFD feeding increases inflammatory and immune pathways in peak lactation mammary glands of mice and increases pup mortality in wild-type (WT) mice compared with dams fed a low-fat diet (LFD). Peripheral serotonin inhibition has been associated with resistance to obesity in male mice fed an HFD. Little is known about the function of Tph1 and how peripheral serotonin affects mammary gland function during pregnancy and lactation. In this study, WT and Tph1-KO models were used to investigate global transcriptomic changes in peak lactation mammary glands when dams were fed either an HFD or LFD. WT and Tph1-KO female mice were assigned to either an LFD or HFD beginning at 3 wk of age (n = 4/group). Dams were euthanized on lactation day 11. Differentially expressed genes (DEGs) were first filtered by adjusted P value (cutoff ≤ 0.05) and fold-change (FC, cutoff ≥2). Genes were further filtered by mean normalized read count with a cutoff 310. We did not observe many differentially expressed genes in WT and Tph1-KO dams fed LFD. However, 3,529 DEGs were observed between WT-HFD and Tph1-KO-HFD mice, including cell cycle regulation and MAPK pathways being significantly enriched. Further research is required to completely understand the physiological significance of our results on peak lactation mammary physiology and the contribution of serotonin.

Maternal stress in Shank3ex4-9 mice increases pup-directed care and alters brain white matter in male offspring.

Gene-environment interactions contribute to the risk for Autism Spectrum Disorder (ASD). Among environmental factors, prenatal exposure to stress may increase the risk for ASD. To examine if there is an interaction between exposure to maternal stress and reduced dosage or loss of Shank3, wild-type (WT), heterozygous (HET) and homozygous (HOM) female mice carrying a deletion of exons four through nine of Shank3 (Shank3ex4-9) were exposed to chronic unpredictable mild stress (CUMS) from prior to conception throughout gestation. This study examined maternal care of these dams and the white matter microstructure in the brains of their adult male offspring. Overall, our findings suggest that maternal exposure to CUMS increased pup-directed care for dams of all three genotypes. Compared to WT and HET dams, HOM dams also exhibited increased maternal care behaviors with increased time spent in the nest and reduced cage exploration, regardless of exposure to CUMS. Diffusion tensor imaging showed higher mean fractional anisotropy in the hippocampal stratum radiatum of WT and HOM male offspring from dams exposed to CUMS and HOM offspring from unexposed dams, compared to WT male offspring from unexposed dams. These data support that CUMS in Shank3-mutant dams results in subtle maternal care alterations and long-lasting changes in the white matter of the hippocampus of their offspring.

Impact of Early Life Antibiotic Exposure and Neonatal Hyperoxia on the Murine Microbiome and Lung Injury

Cross talk between the intestinal microbiome and the lung and its role in lung health remains unknown. Perinatal exposure to antibiotics disrupts the neonatal microbiome and may have an impact on the preterm lung. We hypothesized that perinatal antibiotic exposure leads to long-term intestinal dysbiosis and increased alveolar simplification in a murine hyperoxia model. Pregnant C57BL/6 wild type dams and neonatal mice were treated with antibiotics before and/or immediately after delivery. Control mice received phosphate-buffered saline (PBS). Neonatal mice were exposed to 95% oxygen for 4 days or room air. Microbiome analysis was performed using 16S rRNA gene sequencing. Pulmonary alveolarization and vascularization were analyzed at postnatal day (PND) 21. Perinatal antibiotic exposure modified intestinal beta diversity but not alpha diversity in neonatal mice. Neonatal hyperoxia exposure altered intestinal beta diversity and relative abundance of commensal bacteria in antibiotic treated mice. Hyperoxia disrupted pulmonary alveolarization and vascularization at PND 21; however, there were no differences in the degree of lung injury in antibiotic treated mice compared to vehicle treated controls. Our study suggests that exposure to both hyperoxia and antibiotics early in life may cause long-term alterations in the intestinal microbiome, but intestinal dysbiosis may not significantly influence neonatal hyperoxic lung injury.

N-Acetylcysteine Resolves Placental Inflammatory-Vasculopathic Changes in Mice Consuming a High-Fat Diet

The mechanism by which poor maternal nutrition can affect the long-term health of offspring is poorly understood. In mice, we previously found that maternal high-fat diet (HFD) exposure results in reduced fetal growth regardless of maternal genotype. We tested our hypothesis that maternal HFD-induced inflammation contributes to metabolic disease susceptibility of the offspring via alterations in the placenta. The effect of maternal genotype, diet, and treatment with the anti-inflammatory compound N-acetylcysteine (NAC) on placental morphologic features was investigated. Placentas from wild-type dams maintained on a HFD but not those heterozygous (+/-) for Glut4 (Slc2a4) on the same diet had an increase in decidual inflammation and vasculopathy occurring together. NAC administration resulted in amelioration of HFD-induced decidual vasculopathy independent of offspring genotype and sex. Consistent with these morphologic improvements, placentas from HFD dams treated with NAC had decreased mRNA and immunostaining of IL-1β and monocyte chemoattractant protein-1, decreased mRNA of inflammatory genes, and increased mRNA of Vegfa. These results strongly suggest consumption of an HFD results in vascular changes in placenta reflected by alterations in expression of pivotal vascular developmental markers and inflammatory genes all of which are ameliorated by NAC. These placental changes play a key role in the increased programed metabolic disease of HFD-exposed offspring.

The Role of Beta-carotene Metabolism in Maternal Cardiac Remodeling: Findings in Mice Lacking Beta-carotene 9′,10′-oxygenase (BCO2) (FS06-04-19)

ObjectivesHigh intake of fruits and vegetables, main vitamin A sources, is associated with improved cardiac function. β-carotene, the most abundant dietary precursor of vitamin A, is cleaved by β-carotene 15,15′-oxygenase (BCO1) and β-carotene 9′,10′-oxygenase (BCO2). However, BCO2 is the only β-carotene cleavage enzyme expressed in adult hearts. Cardiac mRNA levels of Bco2 are elevated at mid-gestation in wild-type (WT) mice when the heart is hypertrophic. In the absence of BCO2 (Bco2-/- mice) the maternal heart fails to enlarge. Therefore, we aim to elucidate the role of BCO2 in maternal cardiac hypertrophy and to determine if metabolic pathways in the heart are disrupted by loss of BCO2. We hypothesize that BCO2 contributes to maternal cardiac hypertrophy by affecting homeostasis of RA, the active form of vitamin A. MethodsAge matched WT and Bco2-/- (KO) mice raised on a chow diet were sacrificed at 14.5 days pregnant. Cardiac mRNA and protein expression of retinoid and lipid regulatory genes were measured. HPLC and LC/MS detected cardiac retinoids (vitamin A and its derivatives) levels. ResultsPregnancy (mid-gestation) is associated with cardiac RA deficiency in WT dams. KO mice already showed cardiac RA deficiency pre-pregnancy. KO female mice have reduced PDK4 mRNA expression and enhanced PDH activity (phosphorylation) in the heart, that is reversed earlier, at mid-gestation. KO mice have increased cardiac Glut1 mRNA expression and reduced triglyceride levels. Lipid regulatory genes such as Pgc1a and Scd1 are increased at mid-pregnancy in the heart of WT dams but not in KO mothers. ConclusionsOur data indicates that RA may be involved in modulating the cardiac hypertrophy of pregnancy. Dysregulation of RA homeostasis in the heart of KO females results in metabolic adaptations that makes the heart of the non-pregnant females preferentially dependent on glucose as an energy source. During pregnancy retinoic deficiency in the KO heart may induce an earlier attenuation of PDH activity, facilitating utilization of fat over glucose as energy substrate. These and other findings support the hypothesis of a crucial role of BCO2 in regulating heart hypertrophy, at least in females. Funding SourcesNIH/NHLBI F31.

Maternal Dicer Knockout Impairs Growth and Gut Health in Nursing Wild-type Pups in Mice (P11-137-19)

ObjectivesBackground: Dicer catalyzes microRNA biogenesis. Administration of tamoxifen to heterozygous Dicer conditional knockout (KO) mice (Dicer + /-) deletes one Dicer allele (Dicer KO). Nanoparticles (exosomes) transfer microRNAs from donor cells to recipient cells. Humans absorb exosomes and microRNA cargos from milk. MicroRNAs regulate the expression of 60% of mammalian genes.Objectives: Assess whether the loss of microRNA biogenesis in lactating Dicer KO dams impairs growth and gut health in nursing wild-type pups. MethodsNewborn pups from wild-type (WT) C57BL/6 breeders were fostered to synchronized Dicer + /- dams. Dams were injected with tamoxifen or vehicle (control). Additional controls included WT pups fostered to WT dams injected with tamoxifen or vehicle. Body weight gain and milk consumption were measured at timed intervals up to 21 days when pups were euthanized. Body fat accretion was assessed using NMR. Sections of the gastrointestinal tract were evaluated by histology analyses. The unpaired t-test was used for statistical analysis (tamoxifen vs. vehicle); P < 0.05 was considered statistically significant. ResultsBody weight and fat accretion in male and female pups nursed by Dicer KO dams was significantly lower than in vehicle controls at age 3 weeks (Figs. 1 and 2). Histology assessment revealed villous atrophy in the small intestine from pups nursed by Dicer KO dams compared to vehicle controls at age 3 weeks (Figs. 3–5). Tamoxifen administration did not elicit phenotypes in wild-type pups nursed by wild-type dams (not shown). Milk consumption was not significantly different between pups nursed by Dicer KO dams and controls (not shown). ConclusionsLoss of microRNA biogenesis in lactating Dicer KO dams impairs growth and gut health in nursing wild-type pups. The quality of milk secreted by Dicer KO dams remains to be assessed. Funding SourcesNIFA, NIH, Gerber Foundation, Gates Foundation, PureTech, Inc. and USDA Hatch and Multistate. J.Z. is a consultant for PureTech. Supporting Tables, Images and/or Graphs▪▪▪▪

Mania- and anxiety-like behavior and impaired maternal care in female diacylglycerol kinase eta and iota double knockout mice.

Genome-wide association studies linked diacylglycerol kinase eta and iota to mood disorders, including bipolar disorder and schizophrenia, and both genes are expressed throughout the brain. Here, we generated and behaviorally characterized female mice lacking Dgkh alone, Dgki alone, and double Dgkh/Dgki-knockout (dKO) mice. We found that fewer than 30% of newborn pups raised by dKO females survived to weaning, while over 85% of pups survived to weaning when raised by wild-type (WT) females. Poor survival under the care of dKO mothers was unrelated to pup genotype. Moreover, pups from dKO dams survived when fostered by WT dams, suggesting the poor survival rate of dKO-raised litters was related to impaired maternal care by dKO dams. Nest building was similar between WT and dKO dams; however, some dKO females failed to retrieve any pups in a retrieval assay. Pups raised by dKO dams had smaller or absent milk spots and reduced weight, indicative of impaired nursing. Unlike WT females, postpartum dKO females showed erratic, panicked responses to cage disturbances. Virgin dKO females showed behavioral signs of anxiety and mania, which were not seen in mice lacking either Dgkh or Dgki alone. Our research indicates that combined deletion of Dgkh and Dgki impairs maternal behavior in the early postpartum period, and suggests female dKO mice model symptoms of mania and anxiety.

SAT-226 Maternal Adiponectin Overexpression Decreases Fetal Growth and Alters Metabolic Functions

: Obese women who develop gestational diabetes show lower adiponectin levels across pregnancy than obese euglycaemic women suggesting that obese women with low adiponectin levels have an impaired capacity to handle the metabolic changes during pregnancy. Adiponectin acts on the placenta during pregnancy; this fact allows for the interesting possibility that adiponectin can exert endocrine effects on the developing fetus. The aim here was to investigate how adiponectin affects cellular and molecular mechanisms regulating fetal growth and metabolic functions during pregnancy. Wild type (wt) and adiponectin transgenic (APNtg) mice were fed normal chow or a high fat/high sucrose (HF/HS) diet for 8 weeks before mating with wt males. Fetus were dissected on pregnancy day 18.5. Maternal adiponectin overexpression decreased fetal body weight in dams on normal chow, and even more in dams on HF/HS diet. There was no difference in fasting glucose between the groups on normal chow and HF/HS diet, but APNtg animals had lower fasting glucose levels compared to wt. We also investigated the role of adiponectin on lipid uptake. Wild type dams fed HF/HS diet had decreased levels of circulating triglycerides but increased levels of liver triglycerides. However, APNtg dams were partly protected against the development of fatty liver confirming previous studies. In contrast, liver triglyceride levels were increased in fetuses from APNtg dams on HF/HS diet, suggesting an increased transport of lipids to the developing fetuses. To further investigate placenta function, we measured the expression of genes involved in placental nutrient transport. We found an increased expression of lipoprotein lipase (LPL), while IRS-1 expression was decreased in placentas from APNtg fetuses regardless of the dam’s genotype or diet. Adiponectin has previously been shown to decrease nutrient transport across placenta, with the assumption that the main nutrient transport is glucose. Our data opens up for the possibility that adiponectin may increases lipid delivery to the fetus. Sources of research support: Magnus Bergvall Foundation, Åke Wiberg Foundation, Hjalmar Svensson Foundation, Adlerbert Research Foundation, and Royal Society of Arts and Sciences in Gothenburg

OR12-1 Leptin Is Transported across the Mouse Placenta

The placenta is critical for mother-fetus physiological communication. It is particularly sensitive to changes in the nutritional and hormonal environments and can contribute to the programming of metabolic syndrome. Recent data, including from our laboratory, showed that the hormone leptin influences brain development during fetal life and mediates lifelong metabolic regulations. However, despite the documented capacity of the placenta in transporting various maternally-derived molecules, it is still unclear whether the placenta contributes to circulating fetal leptin. Here, we report that high levels of leptin receptor mRNA were detected in the mouse placenta during mid-gestation, particularly in trophoblast giant cells of the labyrinth zone, where mother-fetus exchanges take place. Remarkably, the main leptin receptor isoform expressed in the placenta was LepRa, which is critical for transporting leptin across cellular barriers. In addition, we found that leptin-deficient (ob/ob) embryos raised by heterozygous dams have significant levels of circulating leptin. Since the mouse placenta does not express leptin mRNA, this data support the idea that fetal leptin is maternally-derived. To further examine leptin transport from mother to fetus, fluorescently labeled bioactive leptin was injected through the tail vein of wild-type dams. Fluorescence was detected not only in the placenta but also in embryo including the hypothalamus, liver and pancreas as early as one minute after injection. In addition, maternally-injected leptin induces STAT3 activation in the placenta and various organs, including the CNS, of the embryo. We are currently examining whether this trans-placental leptin transport is altered in a context of maternal obesity. Taken together, our data provide direct evidence that maternal leptin is transported to the fetus across the mouse placenta.

Regulatory effects of brown adipose tissue thermogenesis on maternal metabolic adaptation, placental efficiency, and fetal growth in mice.

To determine the role of UCP1-mediated thermogenesis in controlling maternal metabolic adaptation to pregnancy, energy metabolism of C57BL/6 wild-type (WT) and Ucp1 gene knockout ( Ucp1-/-) mice was studied during pregnancy. With the progression of pregnancy, maternal energy expenditure rates (EERs), expression of UCP1, and core body temperature steadily declined in WT dams. Despite no significant alterations in core body temperature and weight gain during pregnancy, Ucp1-/- dams exhibited lower rates in EER decline. High-fat (HF) feeding not only robustly increased maternal UCP1 expression and core body temperature but also abolished gestation-suppressed EER in WT dams. However, HF-increased EERs were significantly attenuated in Ucp1-/- dams. Significantly increased fetal body weights and fetal/placental weight ratio were detected in fetuses from Ucp1-/- dams compared with fetuses from WT dams. Markedly increased expression levels of glucose transporter 1 and amino acid transporters were also observed in placentas from Ucp1-/- dams. Furthermore, blood glucose concentrations of fetuses from Ucp1-/- dams were significantly higher than those of fetuses from WT dams, indicating that maternal UCP1 has an inhibitory effect on placental efficiency and fetal growth. Taken all together, this study demonstrated that maternal brown adipose tissue plays an important role in controlling maternal metabolic adaptation and placental nutrient transport.

Milk osteopontin promotes brain development by up-regulating osteopontin in the brain in early life.

Osteopontin (OPN) is a pleiotropic protein and is abundantly present in milk. Its functions include immune modulation and cellular proliferation and differentiation. OPN is highly expressed in the brain. We investigated the effects of milk-derived OPN on brain development of mouse pups. Wild-type (WT) dams producing OPN+ milk and OPN knockout (KO) dams producing OPN- milk nursed WT pups (OPN+/+), yielding 2 pup treatment groups, OPN+ OPN+/+ and OPN- OPN+/+, for comparison. Preliminary studies supported use of this model by showing high concentrations of OPN in milk of WT dams and no OPN in milk of OPN KO dams, and production of similar amounts of milk by WT and KO dams. The ability of ingested milk OPN to enter the brain was revealed by appearance of orally gavaged [125I]-labeled and antibody-probed milk OPN in brains of pups. Brain OPN mRNA levels were similar in both nursed groups, but the brain OPN protein level was significantly lower in the OPN- OPN+/+ group at postnatal days 6 and 8. Behavior tests showed impaired memory and learning ability in OPN- OPN+/+ pups. In addition, our study revealed increased expression of myelination-related proteins and elevated proliferation and differentiation of NG-2 glia into oligodendrocytes in the brain of OPN+ OPN+/+ pups, accompanied by increased activation of ERK-1/2 and PI3K/Akt signaling. We concluded that milk OPN can play an important role in brain development and behavior in infancy by promoting myelination.-Jiang, R., Prell, C., Lönnerdal, B. Milk osteopontin promotes brain development by up-regulating osteopontin in the brain in early life.

Abstract P316: Effects of Paternal Contribution of a Null Allele of Regulator of G Protein Signaling-2 ( Rgs2 ) upon the Placental Transcriptome of C57BL/6J Mice

Preeclampsia (PreE), a cardiovascular disorder of pregnancy, remains a major cause of maternal and fetal mortality worldwide. The early etiology of the disorder is unclear, though increased G protein-coupled receptor signaling has been implicated. Regulator of G protein Signaling-2 (RGS2) is a negative regulator of G protein signaling, and mutations of RGS2 have been associated with increased risk for PreE in humans. Our ongoing work has demonstrated that breeding wildtype C57BL/6J dams with Rgs2 -deficient ( Rgs2 -KO; B6.129P2- Rgs2 tm1Dgen /Mmnc) sires is sufficient to cause ~50% reduced placental expression of Rgs2 mRNA, and to induce hypertension, renal and placental morphological phenotypes typical of PreE in the wildtype dams. To test the hypothesis that reduced RGS2 (via paternal contribution of an Rgs2 -null allele) is sufficient to cause molecular changes within the placenta that are typical of PreE, we performed RNA sequencing on placentas collected on gestational day 12.5 from C57BL/6J dams mated with either Rgs2 -KO sires or wildtype littermates of these sires. RNA was isolated and sequenced using an Illumina HiSeq 4000. Transcript counts were quantified and aligned to the mouse genome (UCSC genome browser) using Kallisto, followed by differential gene expression analysis using DEseq2 (FDR = 0.1). 726 genes were differentially expressed (479 up, 247 down), including some ( Hsd11b2 (up), Adm (down)), that are similarly changed in human PreE placenta (PMID: 28618048 &amp; 26268791). Ingenuity Pathway Analysis indicated that reduced placental Rgs2 expression was associated with mitochondrial dysfunction, unfolded protein response, and oxidative stress (all p &lt; 1e-3), which also parallels findings in human PreE placenta. Collectively these data support the conclusion that reduced Rgs2 expression in the feto-placental unit is sufficient to induce transcriptomic changes within the placenta that are typical of PreE.

Maternal care boosted by paternal imprinting in mammals.

In mammals, mothers are the primary caregiver, programmed, in part, by hormones produced during pregnancy. High-quality maternal care is essential for the survival and lifelong health of offspring. We previously showed that the paternally silenced imprinted gene pleckstrin homology-like domain family A member 2 (Phlda2) functions to negatively regulate a single lineage in the mouse placenta called the spongiotrophoblast, a major source of hormones in pregnancy. Consequently, the offspring’s Phlda2 gene dosage may influence the quality of care provided by the mother. Here, we show that wild-type (WT) female mice exposed to offspring with three different doses of the maternally expressed Phlda2 gene—two active alleles, one active allele (the extant state), and loss of function—show changes in the maternal hypothalamus and hippocampus during pregnancy, regions important for maternal-care behaviour. After birth, WT dams exposed in utero to offspring with the highest Phlda2 dose exhibit decreased nursing and grooming of pups and increased focus on nest building. Conversely, ‘paternalised’ dams, exposed to the lowest Phlda2 dose, showed increased nurturing of their pups, increased self-directed behaviour, and a decreased focus on nest building, behaviour that was robustly maintained in the absence of genetically modified pups. This work raises the intriguing possibility that imprinting of Phlda2 contributed to increased maternal care during the evolution of mammals.