Targeted placental deletion of OGT recapitulates the prenatal stress phenotype including hypothalamic mitochondrial dysfunction

Christopher L. Howerton; Tracy L. Bale

doi:10.1073/pnas.1401203111

Edited* by Bruce S. McEwen, The Rockefeller University, New York, NY, and approved May 19, 2014 (received for review January 22, 2014)

Significance

Early prenatal stress (EPS), a key risk factor for neurodevelopmental disorders, produces a marked reduction in O-GlcNAc transferase (OGT) in male placental tissue. In our current work, we determined that a targeted reduction of placental OGT recapitulates key features of EPS, including hypothalamic–pituitary–adrenal stress axis dysregulation, reduced postpubertal growth, and hypothalamic mitochondrial dysfunction. These results confirm that OGT serves as a key placental biomarker and functions as an important mediator of the maternal changes occurring in response to stress.

Abstract

Maternal stress is a key risk factor in neurodevelopmental disorders, which often have a sex bias in severity and prevalence. We previously identified O-GlcNAc transferase (OGT) as a placental biomarker in our mouse model of early prenatal stress (EPS), where OGT levels were lower in male compared with female tissue and were further decreased following maternal stress. However, the function of placental OGT in programming the developing brain has not been determined. Therefore, we generated a transgenic mouse with targeted placental disruption of Ogt (Pl-OGT) and examined offspring for recapitulation of the adult EPS phenotype. Pl-OGT hemizygous and EPS male placentas showed similar robust changes in gene expression patterns suggestive of an altered ability to respond to endocrine and inflammatory signals, supporting placental OGT as an important mediator of EPS effects. ChIP-Seq for the O-GlcNAc mark identified the 17 beta hydroxysteroid dehydrogenase-3 (Hsd17b3) locus in male EPS placentas, which correlated with a reduction in Hsd17b3 expression and concordant reduced testosterone conversion. Remarkably, Pl-OGT adult offspring had reduced body weights and elevated hypothalamic–pituitary–adrenal stress axis responsivity, recapitulating phenotypes previously reported for EPS males. Further, hypothalamic microarray gene-set enrichment analyses identified reduced mitochondrial function in both Pl-OGT and EPS males. Cytochrome c oxidase activity assays verified this finding, linking reduced placental OGT with critical brain programming. Together, these studies confirm OGT as in important placental biomarker of maternal stress and demonstrate the profound impact a single placental gene has on long-term metabolic and neurodevelopmental programming that may be related to an increased risk for neurodevelopmental disorders.

Maternal stress early in gestation has been identified as a risk factor for neurodevelopmental disorders, including autism spectrum disorders and schizophrenia. Despite strong epidemiological evidence, there remains a lack of specific mechanisms of disease development or predictive biomarkers of disease risk. Examination of the placenta is a promising avenue to find these mechanisms and biomarkers because of features of its biology and accessibility for diagnostic purposes postparturition. The placenta is uniquely positioned at the interface between the maternal and fetal compartments and is rapidly developing during the period of gestation at which maternal stress has been identified to increase disease risk to the offspring (1 ⇓⇓⇓–5). We previously identified a reduction in O-GlcNAc transferase (OGT), an important O-glycotransferase enzyme that plays a critical role in regulation of gene expression through chromatin remodeling, in male placentas following early prenatal stress (EPS) (4). Placental OGT is basally lower in males due to its X-linkage and escaping of X-inactivation in the placenta and further reduced in our mouse model of EPS. Additionally, we established that a targeted reduction of placental OGT using transgenic mouse lines could significantly alter early hypothalamic brain development, supporting an important link between placental OGT and neurodevelopment.

Many of the programmatic changes produced in our EPS mouse model are endophenotypes of neurodevelopmental disorders, including stress dysregulation, cognitive dysfunction, and hypothalamic dysregulation of growth and development (4, 6 ⇓⇓⇓–10). These outcomes support the critical communication from the placenta to the fetus related to the bioenergetic or metabolic environment that then programs the hypothalamus, the brain’s endocrine and metabolic regulatory center. To examine the potential function of OGT in the placenta and its downstream gene targets involved in transmission of the EPS phenotype, we used genetic and epigenetic methodologies to identify the functional intersection between a reduction in OGT and the effects of EPS in the placenta. Based on our hypothesis related to the energetic reprogramming of the hypothalamus resulting from reduced placenta OGT, adult mice from placental-specific OGT (Pl-OGT) hemizygous and homozygous knockout were examined for a recapitulation of an EPS phenotype.

Results

Effects of OGT Complement and EPS on Placental Gene Expression.

To identify placental genes altered by a targeted reduction of placental OGT, we used a genome-wide microarray analysis. Gene expression was evaluated at embryonic day (E)12.5, because this gestational time point is a stage of development with a fully differentiated placenta and has been previously characterized to have significant differences in gene expression of OGT in response to EPS (4). Differential expression analyses [false-discovery rate (FDR) < 0.05] found 485 genes different between placentas with reduced OGT (X^OGT-/X^WT) and WT (X^WT/X^WT) placentas. Of these genes, 277 were down-regulated and 217 were up-regulated in X^OGT-/X^WT (Dataset S1). Functional annotation analyses reveal that the genes down-regulated with reduced placental OGT form five functional groupings: hormone/steroid biosynthesis, anti-inflammatory signaling, amine metabolism, glucagon signaling, and NMDA receptor signaling. Similarly, genes up-regulated with reduced placental OGT were categorized into five functional groupings: protein folding, RNA polymerase I activity, RNA polymerase II activity, mRNA transport, and mitotic cell cycle.

To determine genes and pathways common to both reduced placental OGT and EPS, we performed a similar analysis comparing control placentas to EPS placentas at E12.5. There were 284 genes (both up-regulated and down-regulated; FDR < 0.05; Dataset S1) perturbed by EPS; direct comparison between the genes perturbed by reduced OGT and EPS reveal that only nine individual transcripts are dysregulated in the same direction. Despite the relatively few individual transcripts that share a similar pattern, functional annotation analyses that combine genes significantly changed in at least one of the four conditions (X^OGT-/X^WT, X^WT/X^WT, control and EPS) suggest common regulation of function. The analyses segregate the gene functions into two control and X^WT/X^WT groups (response to endocrine signals and anti-inflammatory signaling; Dataset S1) and two EPS and X^OGT-/X^WT groups (chromatin regulation and RNA/DNA replication and cell cycle; Dataset S1).

To test whether OGT is directly involved in the transcription of genes perturbed both by reduced placental OGT and EPS, we first determined the transcripts most strongly linked by reducing the FDR to 0.01; this resulted in two genes, cold shock domain-containing protein C2 (down-regulated in X^OGT-/X^WT and EPS) and tRNA methyltransferase 11 (up-regulated in X^OGT-/X^WT and EPS). We then compared these genes’ expression with OGT expression in control and EPS placentas. Trmt11 expression was negatively correlated with OGT expression (Fig. S1A), but no relationship was found between Csdc2 and OGT expression (Fig. S1B). Given that Trmt11 expression is elevated in both X^OGT-/X^WT and EPS placentas, and there is a negative correlation between OGT expression and Trmt11 expression, we hypothesized that OGT may be directly involved in the suppression of its transcription by modifying the locus with the posttranslational mark, O-GlcNAc. To test this hypothesis, we performed a ChIP-qPCR experiment in control and EPS placentas. O-GlcNAc patterns were higher in control than EPS placentas upstream of the transcription start site (Fig. S1C) but not downstream of the transcription start site.

Effects of Sex and EPS on O-GlcNAc and H3K4me3 Patterns in the Placenta.

To further investigate the role of OGT in placental chromatin regulation, we performed a genome-wide ChIP-seq experiment at E12.5 comparing control and EPS placentas. For comparison purposes, we immunoprecipitated for both O-GlcNAc, a putative transcriptional repressive mark, and H3K4me3, a permissive chromatin mark. In control placentas, there were 18,676 O-GlcNAc peaks (over background; P < 0.05; Fig. 1A) and 64,935 H3K4me3 peaks. In EPS placentas, there was very little change in H3K4me3 abundance compared with control (57,917 peaks), but O-GlcNAc peaks were reduced by over half (7,887 peaks); these results are consistent with the previously reported reduction of OGT expression in EPS placentas (4). Irrespective of abundance, O-GlcNAc and H3K4me3 marks were consistently concentrated near the transcription start site of protein coding genes (∼90% of each mark within 10,000 bp of a transcription start site; Fig. 1A), suggesting that O-GlcNAc is related to similar regulatory processes of gene transcription as H3K4me3.

Fig. 1.

EPS and genetically reduced OGT impacts placental biology. (A) Distribution of placental ChIP-seq peaks by chromatin modification and treatment. There is a marked reduction of O-GlcNAc (OG), but not H3K4me3 (H3), peaks with EPS. (B) Effect of chromatin state on placental gene expression by treatment. In control placentas, O-GlcNAc synergistically increases expression with H3K4me3, but the opposite is true in EPS placentas. (C) Hsd17b3, the enzyme that catalyzes the conversion of androstenedione to testosterone, has reduced O-GlcNAc occupancy at its locus and a marked reduction in gene expression in EPS placentas compared with control (C). (D) The reduction of Hsd17b3 expression in EPS placentas is correlated with an increase in androstenedione and reduction in testosterone. *Significant difference (by confidence interval inspection) between the groups.

Locus-specific examination of O-GlcNAc peaks in control and EPS placentas reveal there is a marked difference in gene function of loci modified by this mark between these groups. Control placental O-GlcNAc–associated loci are enriched for 11 functional annotations (FDR over background, <0.05; Dataset S2). Notably, over 20% of these loci involve vasculature development and over 30% involve extracellular signal transduction. EPS placental associated loci are enriched for only seven functional annotations (FDR < 0.05; Dataset S2). Of these loci, over 44% involve mitosis and less than 11% involve extracellular signal transduction. To examine the effects of chromatin state of a locus on its gene expression, we fit linear models to microarray expression values that included treatment and presence of an H3K4me3 and/or O-GlcNAc peak at that locus (Fig. 1B). Unsurprisingly, a permissive H3K4me3 peak increased gene expression irrespective of treatment. An O-GlcNac peak on its own had no detectable effect on gene expression in either treatment. Interestingly, having both marks at a locus in control placentas increased gene expression further than H3K4me3 alone, whereas in EPS placentas, the opposite was true. Together, these data suggest that O-GlcNAc may affect gene expression by interacting with histone modifications and further that the nature of this interaction is treatment-dependent.

To determine any loci specifically involved in the male specific effects of EPS mediated by placental chromatin O-GlcNAc, we fit a generalized linear model to peak counts that included treatment, sex, and an interaction between the two predictors. We reasoned that any locus that has differential chromatin O-GlcNAc between male control and EPS placentas with a FDR of <0.05 and a log-fold change of at least 2 would represent a biologically meaningful difference based upon our previous ChIP-qPCR result using O-GlcNAc. Only one locus met these criteria, Hsd17b3, the enzyme that catalyzes the conversion of androstenedione to testosterone, with much less O-GlcNAc at this locus in EPS placentas (Fig. 1C). Expression levels of Hsd17b3 were also much lower EPS placentas than control (Fig. 1C). Functionally, these changes in O-GlcNAc and expression resulted in both higher androstenedione and lower testosterone in EPS placentas compared with controls (Fig. 1D). Together, these data suggest that O-GlcNAc may in part regulate EPS-induced changes in Hsd17b3 expression in the placenta and further that this results in a lower prenatal testosterone production in EPS male placentas.

Effects of Reduced Placental OGT on Growth, Behavior, and Hypothalamic Development.

To determine any long-term and programmatic changes resulting from reduced placental OGT, we compared X^OGT-/Y, X^OGT-/X^WT, X^OGT-/X^OGT- to their same sex and WT littermates (X^WT/Y and X^WT/X^WT) from parturition to adulthood. No detectable differences between genotypes were identified for either anogenital distance (an early sign of sexual differentiation; P > 0.05) or body weights (P > 0.05; Fig. 2 A and B) before 5 wk of age. At 5 wk of age and continuing through 11 wk of age, X^OGT-/Y had lower weights than their WT littermates (Fig. 2A), and interestingly in females, there is a dose–response to placental OGT for body weights with X^OGT-/X^OGT- < X^OGT-/X^WT < X^WT/X^WT (Fig. 2B). Mice with reduced placental OGT also had reduced gonadal fat pad weights and shorter tibial lengths than their WT littermates (Fig. 2 C and D), indicating a shorter and leaner phenotype (Fig. 2E). Behaviorally, there was no difference between genotypes in anxiety-like behavior (as measured by time in the light of the light–dark box in Fig. 2F). Response to a restraint stress was affected by levels of placental OGT: male X^OGT-/Y had an increased hypothalamic–pituitary–adrenal (HPA) response compared with WT littermates (Fig. 2G), whereas X^OGT-/X^OGT- and X^OGT-/X^WT both had a decreased HPA response compared with WT littermates (Fig. 2H).

Fig. 2.

Genetically reduced placental OGT recapitulates key features of the EPS phenotype. Both male (A) and female (B) mice that had genetically reduced placental OGT have a reduced bodyweights after weaning at 4 wk of age compared with WT littermates (n = 8 per genotype). There is a dose–response in this growth retardation with respect to the zygosity of the Ogt locus, observable in the female mice (B). (C and D) Mice with reduced placental OGT also had lower adiposity (as measured by gonadal fat pad weight) (C) and were shorter (as measured by tibial length) (D) compared with WT littermates. (E) Photograph of all possible genotypes of mice (X^OGT-/Y, X^WT/Y, X^OGT-/X^OGT-, X^OGT-/X^WT, X^WT/X^WT). Qualitative observations confirm a shorter leaner phenotype in adult mice that had genetically reduced placental OGT. (F) Genetically reduced placental OGT had no effect on anxiety-like behavior (as measured by time in the light within the light-dark box task; n = 8 per genotype). (G and H) Both male (G) and female (H) mice that had genetically reduced placental OGT have a dysregulated HPA response to a restraint stress compared with WT littermates (n = 8 per genotype). X^OGT-/Y mice have a hyperresponsive HPA (the same direction as EPS males), whereas X^OGT-/X^OGT- and X^OGT-/X^WT have a hyporesponsive HPA (a phenotype not observed in EPS females). *Significant difference (by confidence interval inspection) between the groups.

To determine hypothalamic gene expression changes that could account for the phenotypic differences between X^OGT-/Y and X^WT/Y, we used a genome-wide microarray approach. Differential expression analyses on the individual transcript level reveal over 1,200 genes differentially expressed between X^OGT-/Y and X^WT/Y (Fig. 3A and Dataset S3). Further, there were 20 gene sets differentially expressed between the genotypes, and interestingly all of these gene sets were down-regulated in X^OGT-/Y (Fig. 3B). Of the top 20 gene sets down-regulated in X^OGT-/Y, 6 are closely related to mitochondrial structure and function. Because both reduced placental OGT and EPS result in a reduced body size and HPA hyperresponsivity in males specifically, we also compared hypothalamic gene expression in EPS and control male mice. Gene expression changes were more heterogeneous, with 69 gene sets down-regulated and 5 up-regulated with EPS compared with controls (Dataset S3). Of the top 20 gene sets down-regulated in X^OGT-/Y mice, 4 were also down-regulated in EPS mice (mitochondrial part, metabolic process, carbohydrate metabolic process, and cell redox; Fig. 3B), and all are related to mitochondrial function. Additionally, differential expression analyses determined that of the genes affected by either reduced placental OGT or EPS, 110 were dysregulated in the same direction in the two manipulations (Dataset S3). One such gene, neuropeptide Y (NPY), was significantly decreased both by EPS and reduced placental OGT. Given these results, we hypothesized that the observed dysregulation of hypothalamic functioning in male mice with either reduced placental OGT or that experienced EPS may be in part regulated by altered mitochondrial functioning in this brain region. To test this hypothesis, we measured cytochrome c oxidase (CCO) activity, the rate-limiting step of the electron transport chain, from hypothalamic punches. Both EPS and X^OGT-/Y mice had reduced CCO activity compared with their respective controls (Fig. 3C). Together, these data suggest that there are common hypothalamic gene expression patterns dysregulated by both reduced placental OGT and EPS and further that these expression changes result in altered hypothalamic mitochondrial activity.

Fig. 3.

Reduced placental OGT and EPS result in hypothalamic mitochondrial dysregulation. (A) Heat map of hypothalamic genes with significantly different levels of expression between X^OGT-/Y (n = 6) and XWT/Y mice (n = 5). (B) Differences in gene-set expression values from the comparisons between either X^OGT-/Y and X^WT/Y (white bars; n = 6 or 5, respectively) or male control and EPS (gray bars; n = 6 per group) hypothalamic punches. Both X^OGT-/Y and male EPS had a down-regulation of mitochondrion related gene sets compared with their respective controls. (C) Both EPS and X^OGT-/Y mice had reduced CCO activity at postnatal day 2 compared with their controls (n = 6 per group). CCO is the rate-limiting step of oxidative phosphorylation within the mitochondria. Asterisks represent significant difference (by confidence interval inspection) between the groups.

Discussion

Fetal antecedents, such as maternal stress, are associated with an increased risk for neurodevelopmental disorders, including schizophrenia and autism, often in a sex-specific manner. Our previous work identified reduced OGT as a placental biomarker of male-specific effects of EPS (4). Placental OGT, an important cellular and chromatin regulator, is basally expressed lower in males, and its expression is further reduced by maternal prenatal stress. Additionally, we reported that a targeted reduction of placental OGT resulted in profound disruption in early hypothalamic gene expression patterns. To determine a functional link between placental OGT and transmission of information important in programming changes in the brain, we examined placental gene expression and chromatin regulation patterns, as well as the adult phenotype of placental-specific OGT knockout mice for predictive validity of this EPS biomarker.

OGT broadly affects cellular functioning through the unique posttranslational modification, O-GlcNAc, on serine and threonine residues of over 1,000 different intracellular proteins (11). Many of these protein targets (notably RNA polymerase II, histone deacetylase-2, Tet proteins, and core histone H2B) are part of chromatin structure or directly regulate chromatin confirmation (12 ⇓⇓⇓⇓–17). Therefore, we hypothesized that reduced placental OGT may be a mechanism through which EPS alters placental function. In support of this hypothesis, we found specific gene expression patterns that were common between EPS placentas and placentas from genetically reduced placental OGT. Specifically, we detected a reduction in expression in functional gene sets involved in endocrine and anti-inflammatory signaling and an increase in those genes important in mitosis and RNA/DNA replication and cell cycling, suggesting that a reduction of OGT (either transgenically or by EPS) could account for profound changes in the endocrine function of the placenta and subsequent differences in the relay of important signals to the developing fetus. Similarly, we found by ChIP-Seq analyses that levels of O-GlcNAcylation followed a similar pattern, where the majority of O-GlcNAcylated loci in control tissues involved responses to extracellular signaling, whereas the largest portion of loci identified in EPS placentas were involved with mitosis. Together, these data suggest that normal placentas at this stage of development are fully differentiated and able to respond to environmental conditions, whereas EPS and placentas with reduced OGT may still be progressing through a stage of rapid cellular proliferation. Such outcomes support that maternal stress early in pregnancy promotes a delay in the maturation of the placenta through its effects on OGT, such as by altered epigenetic regulation of key developmental genes by OGT, O-GlcNAc, and its coregulators, including regulation of the recently identified demethylases, Tet1 and Tet2 (13, 18, 19). Delayed placental maturation has been associated with various clinical complications, including placental insufficiency (20), and has also been associated with altered neurodevelopment (21). This may be an evolutionary advantage whereby growth and development are delayed because of a less favorable environment.

OGT has been linked to transcriptional control in both invertebrates and mammals, specifically via modification of RNA polymerase II and various transcription factors at the promoters of genes important for growth, nutrient sensing and immunity (22, 23). Despite this linkage, there has not been a consensus as to the effects this modification has on expression of the associated locus, with some data suggesting a predominantly repressive role (24), whereas others have suggested a more permissive one (25). This likely reflects the complex milieu of proteins involved at the promoters of genes, as well as the dynamic nature of the O-GlcNAc mark (26), and emphasizes the importance of understanding tissue and locus-specific mechanisms. In this study, we found that O-GlcNAc had treatment-specific effects on placental transcription across the entire genome, but locus-specific effects were noted as well. On balance, O-GlcNAc appeared to have little effect on gene expression when the permissive histone mark, H3K4me3, was not detected at the locus. When both O-GlcNAc and H3K4me3 marks were present they appeared to synergistically increase expression of a locus in control placentas but had the opposite effect in EPS placentas. These data suggest that the contributions of placental OGT toward an EPS phenotype likely involve complex and locus specific interactions at the chromatin level that may impact placental function throughout gestation.

Focusing on specific loci that may be predictive of an EPS male phenotype, we identified Hsd17b3, the enzyme that catalyzes the conversion of androstenedione to testosterone. There was a significant reduction of O-GlcNAc at this locus in male EPS placentas compared with control and was further associated with a reduction in expression of the gene. Functionally, as would be predicted from reduced 17βHSD-3, male EPS placental tissue showed a significant increase in androstenedione levels and a significant reduction in testosterone. We have previously reported that EPS results in a dysmasculinization phenotype in first and second generation male offspring, including HPA axis hyperresponsivity, smaller testes, and a shortened anogenital distance (6), and yet the specific mechanism for this reduction in masculinization has not been determined. Fetal testosterone is important in the organization of the sexually dimorphic brain, and de novo placental production of this hormone may be a significant contributor (27 ⇓⇓–30). In conjunction with our data, this suggests that OGT can regulate placental testosterone production, thus providing a functional mechanism for some aspects of the EPS dysmasculinized phenotype.

To determine a potential recapitulation of the EPS phenotype in Pl-OGT offspring to better link changes in placental function with hypothalamic reprogramming, we examined adult offspring for outcomes similar to those we previously reported for EPS mice (4, 6 ⇓⇓⇓–10). Similar to EPS mice, Pl-OGT mice showed slowed growth and weight gain compared to control littermates, resulting in a 10–20% difference in end weights as adults (10). From birth through weaning, however, Pl-OGT mice were normal in these measures, showing no differences in body weights, suggesting that the effect may be driven by growth or gonadal hormone changes around puberty or related to the stress of weaning. Intriguingly, for this phenotype there was a gene dosage effect where in females, hemizygous Pl-OGT mice fell between WT and homozygous Pl-OGT for body-length and weight measurements, supporting the critical impact of tight regulation of OGT levels in the placenta on neurodevelopment.

In addition to the body weight phenotype, mice with reduced placental OGT also had HPA stress axis dysregulation. In recapitulation of male EPS mice, male Pl-OGT mice showed a heightened corticosterone response to a restraint stress but a normal recovery period (6, 9). Although the effect was in the same direction in males as the EPS males, the effect size was modest, suggesting that placental OGT does not account for the entire phenotype found in EPS males. These data also suggest that the effect on the HPA sensitivity in these mice is at the level of the corticotropin-releasing factor neuron, possibly within the paraventricular nucleus as predicted from our array results, rather than the glucocorticoid receptor-mediated feedback in the ventral hippocampus. In contrast, and to our surprise, female hemi-and homozygous Pl-OGT mice showed the opposite effect, with a blunted HPA stress axis, an observation not previously found in female EPS mice. This sex-specific programming by placental OGT is intriguing because it points to a likely involvement of additional placental X- or Y-linked genes in determining adult stress axis programming.

OGT is an important cellular nutrient, stress, and immune sensor (16, 22, 31). In the placenta, OGT appears to provide signals as to an altered environment (nutritive and/or stressors) that shapes hypothalamic circuits involved in long-term programming of energy homeostasis and stress responsivity in a sex-dependent manner. To further examine this, we compared hypothalamic gene expression patterns in adult Pl-OGT male mice. Fitting with our hypothesis, functional gene-set analyses pointed to dramatic changes in genes involved in mitochondrial function and energy metabolism. These results are similar to our previously reported hypothalamic gene expression changes in the neonatal hypothalamus Pl-OGT mice (4). Amazingly, in our comparison with the EPS male hypothalamus, we found a very similar pattern of gene expression, suggesting reduced mitochondrial function. We also examined the microarray data with a gene candidate approach and found a specific reduction in expression of NPY, an important regulator of energy homeostasis and stress responsivity, supporting the hypothesis that dysregulation is at the level of the developing hypothalamus (32). We used a CCO assay as a readout of mitochondria function and compared EPS and Pl-OGT mice. Results confirmed that in both EPS and Pl-OGT mice, hypothalamic mitochondria function was significantly reduced, linking placental OGT again to EPS brain programming. Hypothalamic mitochondrial dysfunction has previously been associated with altered whole-body growth and energy homeostasis, as well as stress-related depressive-like behaviors (33 ⇓⇓⇓⇓–38). Therefore, the observed phenotypes in male EPS and Pl-OGT mice (HPA hyperresponsivity and dysregulated energy homeostasis) appear to be, at least in part, the result of mitochondrial dysfunction programmed by a reduction in placental OGT during gestation.

Using both our EPS mouse model and a transgenic Pl-OGT mouse, these studies have demonstrated that placental gene expression and chromatin structure, subsequent offspring development, and hypothalamic mitochondrial function are all substantially altered by the reduction of one key placental gene, OGT. Importantly, there is a strong association between male biased neurodevelopmental disorders (e.g., autism spectrum disorders and schizophrenia) with metabolic (39, 40) and HPA (41, 42) dysregulation, providing face validity for our findings as to the importance of the tight regulation and functional level of this protein in the placenta. These studies demonstrate a remarkable linkage between placental OGT and programming of the developing hypothalamus capable of producing profound long-term metabolic dysregulation, predictive of neurodevelopmental disorders.

Methods

Animals.

Male C57BL/6J and female 129S1/SvImJ mice were obtained from The Jackson Laboratories and subsequently used as breeding stock to produce C57BL/6J:129S1/SvImJ hybrids (F1 hybrids). F1 hybrid breeding pairs (n = 21) were checked daily at 0700 hours for copulation plugs; 1200 hours of the day that the plug was observed was considered to be E0.5. F1 hybrids were used for the prenatal stress experiments. For the placental specific reduction of OGT (Pl-OGT), double-heterozygous [B6.129-Ogt^tm1Gwh/J (X^OGT/X^WT); B6-CYP19-Cre (P.Cre^+/−)] females (n = 21) were bred to hemizygous [B6.129-Ogt^tm1Gwh/J (X^OGT/Y)/heterozygous (P.Cre^+/−)] males (n = 11), resulting in offspring representing all potential genotypes [female X^WT/X^WT P.Cre⁺ or female X^WT/X^WT P.Cre⁻ (F:WT/WT); female X^OGT/X^WT P.Cre⁺ (F:OGT-/WT); female X^OGT/X^OGT P.Cre⁺ (F:OGT-/OGT-); male X^WT/Y P.Cre⁺ or male X^WT/Y P.Cre⁻ (M:WT/Y); or male X^OGT/Y P.Cre⁺ (M:OGT-/Y)], which were used for analyses. All animal protocols were reviewed and approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania.

EPS.

Administration of chronic variable stress was performed as previously described (9). Dams were randomly assigned to treatment groups to receive stress during days 1–7 of gestation (EPS; n = 13) or to a control (n = 14) nonstressed group. Pregnant mice assigned to the EPS group experienced each of the following stressors (in random order) on a different day of the EPS period: 60 min (beginning at 1300 hours) of fox odor exposure (1:5,000 2,4,5-trimethylthiazole; Acros Organics), 15 min of restraint (beginning at 1300 hours) in a modified 50-mL conical tube, 36 h of constant light, novel noise (White Noise/Nature Sound-Sleep Machine; Brookstone) overnight, three cage changes (at ∼0900, 1200, and 1500 hours) throughout the light cycle, saturated bedding (700 mL, 23 °C water) overnight, and novel object (eight marbles of similar shape and color) exposure overnight. These stressors were selected to be nonhabituating and for not inducing pain or directly influencing maternal food intake, weight gain, or behavior (7).

Additional methods may be found in SI Methods.

Acknowledgments

We thank C. Howard for technical assistance and animal care, G. Leone (The Ohio State University) for the generous donation of the Cyp19-Cre mice, S. Srinivasan and N. Avadhani for technical assistance with the CCO activity assay, and C. Morgan for helpful comments on the manuscript. This work is supported by National Institutes of Health Grants MH091258, MH087597, and MH099910.

Footnotes

¹To whom correspondence should be addressed. E-mail: tbale{at}vet.upenn.edu.

Author contributions: C.L.H. and T.L.B. designed research; C.L.H. performed research; C.L.H. analyzed data; and C.L.H. and T.L.B. wrote the paper.
The authors declare no conflict of interest.
↵*This Direct Submission article had a prearranged editor.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1401203111/-/DCSupplemental.

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(2013) Proteomic characterization of novel histone post-translational modifications. Epigenetics Chromatin 6(1):24.
OpenUrl CrossRef PubMed
↵
1. Deplus R,
2. et al.
(2013) TET2 and TET3 regulate GlcNAcylation and H3K4 methylation through OGT and SET1/COMPASS. EMBO J 32(5):645–655.
OpenUrl Abstract/FREE Full Text
↵
1. Fujiki R,
2. et al.
(2011) GlcNAcylation of histone H2B facilitates its monoubiquitination. Nature 480(7378):557–560.
OpenUrl CrossRef PubMed
↵
1. Hanover JA,
2. Krause MW,
3. Love DC
(2012) Bittersweet memories: Linking metabolism to epigenetics through O-GlcNAcylation. Nat Rev Mol Cell Biol 13(5):312–321.
OpenUrl CrossRef PubMed
↵
1. Hwang S-Y,
2. Hwang J-S,
3. Kim S-Y,
4. Han I-O
(2013) O-GlcNAcylation and p50/p105 binding of c-Rel are dynamically regulated by LPS and glucosamine in BV2 microglia cells. Br J Pharmacol 169(7):1551–1560.
OpenUrl CrossRef PubMed
↵
1. Shi F-T,
2. et al.
(2013) Ten-eleven translocation 1 (Tet1) is regulated by O-linked N-acetylglucosamine transferase (Ogt) for target gene repression in mouse embryonic stem cells. J Biol Chem 288(29):20776–20784.
OpenUrl Abstract/FREE Full Text
↵
1. Chen Q,
2. Chen Y,
3. Bian C,
4. Fujiki R,
5. Yu X
(2013) TET2 promotes histone O-GlcNAcylation during gene transcription. Nature 493(7433):561–564.
OpenUrl CrossRef PubMed
↵
1. Vella P,
2. et al.
(2013) Tet proteins connect the O-linked N-acetylglucosamine transferase Ogt to chromatin in embryonic stem cells. Mol Cell 49(4):645–656.
OpenUrl CrossRef PubMed
↵
1. Sivrioğlu AK,
2. et al.
(2013) Evaluation of the placenta with relative apparent diffusion coefficient and T2 signal intensity analysis. Diagn Interv Radiol 19(6):495–500.
OpenUrl PubMed
↵
1. Harteman JC,
2. et al.
(2013) Placental pathology in full-term infants with hypoxic-ischemic neonatal encephalopathy and association with magnetic resonance imaging pattern of brain injury. J Pediatr 163(4):968.e2–975.e2.
OpenUrl
↵
1. Lewis BA
(2013) O-GlcNAcylation at promoters, nutrient sensors, and transcriptional regulation. Biochim Biophys Acta 1829(11):1202–1206.
OpenUrl
↵
1. Li MD,
2. et al.
(2012) O-GlcNAc transferase is involved in glucocorticoid receptor-mediated transrepression. J Biol Chem 287(16):12904–12912.
OpenUrl Abstract/FREE Full Text
↵
1. Gambetta MC,
2. Oktaba K,
3. Müller J
(2009) Essential role of the glycosyltransferase sxc/Ogt in polycomb repression. Science 325(5936):93–96.
OpenUrl Abstract/FREE Full Text
↵
1. Ramakrishnan P,
2. et al.
(2013) Activation of the transcriptional function of the NF-κB protein c-Rel by O-GlcNAc glycosylation. Sci Signal 6(290):ra75.
OpenUrl Abstract/FREE Full Text
↵
1. Zachara NE,
2. et al.
(2004) Dynamic O-GlcNAc modification of nucleocytoplasmic proteins in response to stress. A survival response of mammalian cells. J Biol Chem 279(29):30133–30142.
OpenUrl Abstract/FREE Full Text
↵
1. Lombardo MV,
2. et al.
(2012) Fetal testosterone influences sexually dimorphic gray matter in the human brain. J Neurosci 32(2):674–680.
OpenUrl Abstract/FREE Full Text
↵
1. Escobar JC,
2. Carr BR
(2011) The protein kinase a pathway regulates CYP17 expression and androgen production in the human placenta. J Clin Endocrinol Metab 96(9):2869–2873.
OpenUrl CrossRef PubMed
↵
1. Escobar JC,
2. Patel SS,
3. Beshay VE,
4. Suzuki T,
5. Carr BR
(2011) The human placenta expresses CYP17 and generates androgens de novo. J Clin Endocrinol Metab 96(5):1385–1392.
OpenUrl CrossRef PubMed
↵
1. Fernández-Guasti A,
2. Fiedler JL,
3. Herrera L,
4. Handa RJ
(2012) Sex, stress, and mood disorders: At the intersection of adrenal and gonadal hormones. Horm Metab Res 44(8):607–618.
OpenUrl CrossRef PubMed
↵
1. Lazarus MB,
2. Nam Y,
3. Jiang J,
4. Sliz P,
5. Walker S
(2011) Structure of human O-GlcNAc transferase and its complex with a peptide substrate. Nature 469(7331):564–567.
OpenUrl CrossRef PubMed
↵
1. Heilig M
(2004) The NPY system in stress, anxiety and depression. Neuropeptides 38(4):213–224.
OpenUrl CrossRef PubMed
↵
1. Theoharides TC
(2013) Is a subtype of autism an allergy of the brain? Clin Ther 35(5):584–591.
OpenUrl CrossRef PubMed
↵
1. Kim MS,
2. Quan W,
3. Lee MS
(2013) Role of hypothalamic autophagy in the control of whole body energy balance. Rev Endocr Metab Disord 14(4):377–386.
OpenUrl CrossRef PubMed
↵
1. Villa RF,
2. Ferrari F,
3. Gorini A
(2012) Energy metabolism of rat cerebral cortex, hypothalamus and hypophysis during ageing. Neuroscience 227:55–66.
OpenUrl CrossRef PubMed
↵
1. Carneiro L,
2. et al.
(2012) Importance of mitochondrial dynamin-related protein 1 in hypothalamic glucose sensitivity in rats. Antioxid Redox Signal 17(3):433–444.
OpenUrl CrossRef PubMed
↵
1. Nazaryan NS,
2. et al.
(2011) Region-specific nitric oxide production in cytosolic and mitochondrial compartments of the rat brain tissues following chronic stress-induced depression-like behavior. Biopolymers Cell 27(5):394–397.
OpenUrl CrossRef
↵
1. Dietrich MO,
2. Liu ZW,
3. Horvath TL
(2013) Mitochondrial dynamics controlled by mitofusins regulate Agrp neuronal activity and diet-induced obesity. Cell 155(1):188–199.
OpenUrl CrossRef PubMed
↵
1. Rossignol DA,
2. Frye RE
(2012) A review of research trends in physiological abnormalities in autism spectrum disorders: Immune dysregulation, inflammation, oxidative stress, mitochondrial dysfunction and environmental toxicant exposures. Mol Psychiatry 17(4):389–401.
OpenUrl CrossRef PubMed
↵
1. Harris LW,
2. et al.
(2013) Schizophrenia: Metabolic aspects of aetiology, diagnosis and future treatment strategies. Psychoneuroendocrinology 38(6):752–766.
OpenUrl CrossRef PubMed
↵
1. Bale TL
(2011) Sex differences in prenatal epigenetic programming of stress pathways. Stress 14(4):348–356.
OpenUrl PubMed
↵
1. Bale TL,
2. et al.
(2010) Early life programming and neurodevelopmental disorders. Biol Psychiatry 68(4):314–319.
OpenUrl CrossRef PubMed

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Biological Sciences
Neuroscience

Proceedings of the National Academy of Sciences: 111 (26)

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[1] ↵
Broad KD,
Keverne EB
(2011) Placental protection of the fetal brain during short-term food deprivation. Proc Natl Acad Sci USA 108(37):15237–15241.
OpenUrl Abstract/FREE Full Text

[2] Broad KD,

[3] Keverne EB

[4] ↵
Rossant J,
Cross JC
(2001) Placental development: Lessons from mouse mutants. Nat Rev Genet 2(7):538–548.
OpenUrl PubMed

[5] Rossant J,

[6] Cross JC

[7] ↵
Rosenfeld CS
(2012) Effects of maternal diet and exposure to bisphenol A on sexually dimorphic responses in conceptuses and offspring. Reprod Domest Anim 47(Suppl 4):23–30.
OpenUrl CrossRef PubMed

[8] Rosenfeld CS

[9] ↵
Howerton CL,
Morgan CP,
Fischer DB,
Bale TL
(2013) O-GlcNAc transferase (OGT) as a placental biomarker of maternal stress and reprogramming of CNS gene transcription in development. Proc Natl Acad Sci USA 110(13):5169–5174.
OpenUrl Abstract/FREE Full Text

[10] Howerton CL,

[11] Morgan CP,

[12] Fischer DB,

[13] Bale TL

[14] ↵
Kapoor A,
Petropoulos S,
Matthews SG
(2008) Fetal programming of hypothalamic-pituitary-adrenal (HPA) axis function and behavior by synthetic glucocorticoids. Brain Res Brain Res Rev 57(2):586–595.
OpenUrl CrossRef PubMed

[15] Kapoor A,

[16] Petropoulos S,

[17] Matthews SG

[18] ↵
Morgan CP,
Bale TL
(2011) Early prenatal stress epigenetically programs dysmasculinization in second-generation offspring via the paternal lineage. J Neurosci 31(33):11748–11755.
OpenUrl Abstract/FREE Full Text

[19] Morgan CP,

[20] Bale TL

[21] ↵
Mueller BR,
Bale TL
(2006) Impact of prenatal stress on long term body weight is dependent on timing and maternal sensitivity. Physiol Behav 88(4-5):605–614.
OpenUrl CrossRef PubMed

[22] Mueller BR,

[23] Bale TL

[24] ↵
Mueller BR,
Bale TL
(2007) Early prenatal stress impact on coping strategies and learning performance is sex dependent. Physiol Behav 91(1):55–65.
OpenUrl CrossRef PubMed

[25] Mueller BR,

[26] Bale TL

[27] ↵
Mueller BR,
Bale TL
(2008) Sex-specific programming of offspring emotionality after stress early in pregnancy. J Neurosci 28(36):9055–9065.
OpenUrl Abstract/FREE Full Text

[28] Mueller BR,

[29] Bale TL

[30] ↵
Pankevich DE,
Mueller BR,
Brockel B,
Bale TL
(2009) Prenatal stress programming of offspring feeding behavior and energy balance begins early in pregnancy. Physiol Behav 98(1-2):94–102.
OpenUrl CrossRef PubMed

[31] Pankevich DE,

[32] Mueller BR,

[33] Brockel B,

[34] Bale TL

[35] ↵
Wang J,
Torii M,
Liu H,
Hart GW,
Hu Z-Z
(2011) dbOGAP - an integrated bioinformatics resource for protein O-GlcNAcylation. BMC Bioinformatics 12(1):91.
OpenUrl CrossRef PubMed

[36] Wang J,

[37] Torii M,

[38] Liu H,

[39] Hart GW,

[40] Hu Z-Z

[41] ↵
Arnaudo AM,
Garcia BA
(2013) Proteomic characterization of novel histone post-translational modifications. Epigenetics Chromatin 6(1):24.
OpenUrl CrossRef PubMed

[42] Arnaudo AM,

[43] Garcia BA

[44] ↵
Deplus R,
et al.
(2013) TET2 and TET3 regulate GlcNAcylation and H3K4 methylation through OGT and SET1/COMPASS. EMBO J 32(5):645–655.
OpenUrl Abstract/FREE Full Text

[45] Deplus R,

[46] et al.

[47] ↵
Fujiki R,
et al.
(2011) GlcNAcylation of histone H2B facilitates its monoubiquitination. Nature 480(7378):557–560.
OpenUrl CrossRef PubMed

[48] Fujiki R,

[49] et al.

[50] ↵
Hanover JA,
Krause MW,
Love DC
(2012) Bittersweet memories: Linking metabolism to epigenetics through O-GlcNAcylation. Nat Rev Mol Cell Biol 13(5):312–321.
OpenUrl CrossRef PubMed

[51] Hanover JA,

[52] Krause MW,

[53] Love DC

[54] ↵
Hwang S-Y,
Hwang J-S,
Kim S-Y,
Han I-O
(2013) O-GlcNAcylation and p50/p105 binding of c-Rel are dynamically regulated by LPS and glucosamine in BV2 microglia cells. Br J Pharmacol 169(7):1551–1560.
OpenUrl CrossRef PubMed

[55] Hwang S-Y,

[56] Hwang J-S,

[57] Kim S-Y,

[58] Han I-O

[59] ↵
Shi F-T,
et al.
(2013) Ten-eleven translocation 1 (Tet1) is regulated by O-linked N-acetylglucosamine transferase (Ogt) for target gene repression in mouse embryonic stem cells. J Biol Chem 288(29):20776–20784.
OpenUrl Abstract/FREE Full Text

[60] Shi F-T,

[61] et al.

[62] ↵
Chen Q,
Chen Y,
Bian C,
Fujiki R,
Yu X
(2013) TET2 promotes histone O-GlcNAcylation during gene transcription. Nature 493(7433):561–564.
OpenUrl CrossRef PubMed

[63] Chen Q,

[64] Chen Y,

[65] Bian C,

[66] Fujiki R,

[67] Yu X

[68] ↵
Vella P,
et al.
(2013) Tet proteins connect the O-linked N-acetylglucosamine transferase Ogt to chromatin in embryonic stem cells. Mol Cell 49(4):645–656.
OpenUrl CrossRef PubMed

[69] Vella P,

[70] et al.

[71] ↵
Sivrioğlu AK,
et al.
(2013) Evaluation of the placenta with relative apparent diffusion coefficient and T2 signal intensity analysis. Diagn Interv Radiol 19(6):495–500.
OpenUrl PubMed

[72] Sivrioğlu AK,

[73] et al.

[74] ↵
Harteman JC,
et al.
(2013) Placental pathology in full-term infants with hypoxic-ischemic neonatal encephalopathy and association with magnetic resonance imaging pattern of brain injury. J Pediatr 163(4):968.e2–975.e2.
OpenUrl

[75] Harteman JC,

[76] et al.

[77] ↵
Lewis BA
(2013) O-GlcNAcylation at promoters, nutrient sensors, and transcriptional regulation. Biochim Biophys Acta 1829(11):1202–1206.
OpenUrl

[78] Lewis BA

[79] ↵
Li MD,
et al.
(2012) O-GlcNAc transferase is involved in glucocorticoid receptor-mediated transrepression. J Biol Chem 287(16):12904–12912.
OpenUrl Abstract/FREE Full Text

[80] Li MD,

[81] et al.

[82] ↵
Gambetta MC,
Oktaba K,
Müller J
(2009) Essential role of the glycosyltransferase sxc/Ogt in polycomb repression. Science 325(5936):93–96.
OpenUrl Abstract/FREE Full Text

[83] Gambetta MC,

[84] Oktaba K,

[85] Müller J

[86] ↵
Ramakrishnan P,
et al.
(2013) Activation of the transcriptional function of the NF-κB protein c-Rel by O-GlcNAc glycosylation. Sci Signal 6(290):ra75.
OpenUrl Abstract/FREE Full Text

[87] Ramakrishnan P,

[88] et al.

[89] ↵
Zachara NE,
et al.
(2004) Dynamic O-GlcNAc modification of nucleocytoplasmic proteins in response to stress. A survival response of mammalian cells. J Biol Chem 279(29):30133–30142.
OpenUrl Abstract/FREE Full Text

[90] Zachara NE,

[91] et al.

[92] ↵
Lombardo MV,
et al.
(2012) Fetal testosterone influences sexually dimorphic gray matter in the human brain. J Neurosci 32(2):674–680.
OpenUrl Abstract/FREE Full Text

[93] Lombardo MV,

[94] et al.

[95] ↵
Escobar JC,
Carr BR
(2011) The protein kinase a pathway regulates CYP17 expression and androgen production in the human placenta. J Clin Endocrinol Metab 96(9):2869–2873.
OpenUrl CrossRef PubMed

[96] Escobar JC,

[97] Carr BR

[98] ↵
Escobar JC,
Patel SS,
Beshay VE,
Suzuki T,
Carr BR
(2011) The human placenta expresses CYP17 and generates androgens de novo. J Clin Endocrinol Metab 96(5):1385–1392.
OpenUrl CrossRef PubMed

[99] Escobar JC,

[100] Patel SS,

[101] Beshay VE,

[102] Suzuki T,

[103] Carr BR

[104] ↵
Fernández-Guasti A,
Fiedler JL,
Herrera L,
Handa RJ
(2012) Sex, stress, and mood disorders: At the intersection of adrenal and gonadal hormones. Horm Metab Res 44(8):607–618.
OpenUrl CrossRef PubMed

[105] Fernández-Guasti A,

[106] Fiedler JL,

[107] Herrera L,

[108] Handa RJ

[109] ↵
Lazarus MB,
Nam Y,
Jiang J,
Sliz P,
Walker S
(2011) Structure of human O-GlcNAc transferase and its complex with a peptide substrate. Nature 469(7331):564–567.
OpenUrl CrossRef PubMed

[110] Lazarus MB,

[111] Nam Y,

[112] Jiang J,

[113] Sliz P,

[114] Walker S

[115] ↵
Heilig M
(2004) The NPY system in stress, anxiety and depression. Neuropeptides 38(4):213–224.
OpenUrl CrossRef PubMed

[116] Heilig M

[117] ↵
Theoharides TC
(2013) Is a subtype of autism an allergy of the brain? Clin Ther 35(5):584–591.
OpenUrl CrossRef PubMed

[118] Theoharides TC

[119] ↵
Kim MS,
Quan W,
Lee MS
(2013) Role of hypothalamic autophagy in the control of whole body energy balance. Rev Endocr Metab Disord 14(4):377–386.
OpenUrl CrossRef PubMed

[120] Kim MS,

[121] Quan W,

[122] Lee MS

[123] ↵
Villa RF,
Ferrari F,
Gorini A
(2012) Energy metabolism of rat cerebral cortex, hypothalamus and hypophysis during ageing. Neuroscience 227:55–66.
OpenUrl CrossRef PubMed

[124] Villa RF,

[125] Ferrari F,

[126] Gorini A

[127] ↵
Carneiro L,
et al.
(2012) Importance of mitochondrial dynamin-related protein 1 in hypothalamic glucose sensitivity in rats. Antioxid Redox Signal 17(3):433–444.
OpenUrl CrossRef PubMed

[128] Carneiro L,

[129] et al.

[130] ↵
Nazaryan NS,
et al.
(2011) Region-specific nitric oxide production in cytosolic and mitochondrial compartments of the rat brain tissues following chronic stress-induced depression-like behavior. Biopolymers Cell 27(5):394–397.
OpenUrl CrossRef

[131] Nazaryan NS,

[132] et al.

[133] ↵
Dietrich MO,
Liu ZW,
Horvath TL
(2013) Mitochondrial dynamics controlled by mitofusins regulate Agrp neuronal activity and diet-induced obesity. Cell 155(1):188–199.
OpenUrl CrossRef PubMed

[134] Dietrich MO,

[135] Liu ZW,

[136] Horvath TL

[137] ↵
Rossignol DA,
Frye RE
(2012) A review of research trends in physiological abnormalities in autism spectrum disorders: Immune dysregulation, inflammation, oxidative stress, mitochondrial dysfunction and environmental toxicant exposures. Mol Psychiatry 17(4):389–401.
OpenUrl CrossRef PubMed

[138] Rossignol DA,

[139] Frye RE

[140] ↵
Harris LW,
et al.
(2013) Schizophrenia: Metabolic aspects of aetiology, diagnosis and future treatment strategies. Psychoneuroendocrinology 38(6):752–766.
OpenUrl CrossRef PubMed

[141] Harris LW,

[142] et al.

[143] ↵
Bale TL
(2011) Sex differences in prenatal epigenetic programming of stress pathways. Stress 14(4):348–356.
OpenUrl PubMed

[144] Bale TL

[145] ↵
Bale TL,
et al.
(2010) Early life programming and neurodevelopmental disorders. Biol Psychiatry 68(4):314–319.
OpenUrl CrossRef PubMed

[146] Bale TL,

[147] et al.

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Targeted placental deletion of OGT recapitulates the prenatal stress phenotype including hypothalamic mitochondrial dysfunction

Significance

Abstract

Results

Effects of OGT Complement and EPS on Placental Gene Expression.

Effects of Sex and EPS on O-GlcNAc and H3K4me3 Patterns in the Placenta.

Effects of Reduced Placental OGT on Growth, Behavior, and Hypothalamic Development.

Discussion

Methods

Animals.

EPS.

Acknowledgments

Footnotes

References

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Search

Significance

Abstract

Results

Effects of OGT Complement and EPS on Placental Gene Expression.

Effects of Sex and EPS on O-GlcNAc and H3K4me3 Patterns in the Placenta.

Effects of Reduced Placental OGT on Growth, Behavior, and Hypothalamic Development.

Discussion

Methods

Animals.

EPS.

Acknowledgments

Footnotes

References

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Article Classifications

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