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O-GlcNAc transferase (OGT) as a placental biomarkerof maternal
stress and reprogramming of CNS genetranscription in
developmentChristopher L. Howerton, Christopher P. Morgan, David B.
Fischer, and Tracy L. Bale1
Department of Animal Biology, School of Veterinary Medicine,
University of Pennsylvania, Philadelphia, PA 19104
Edited by Bruce S. McEwen, The Rockefeller University, New York,
NY, and approved February 6, 2013 (received for review January 4,
2013)
Maternal stress is a key risk factor for neurodevelopmental
disor-ders, including schizophrenia and autism, which often exhibit
a sexbias in rates of presentation, age of onset, and symptom
severity.The placenta is an endocrine tissue that functions as an
importantmediator in responding to perturbations in the
intrauterine en-vironment and is accessible for diagnostic
purposes, potentially pro-viding biomarkers predictive of disease.
Therefore, we have used agenome-wide array approach to screen
placental expression acrosspregnancy for gene candidates that are
sex-biased and stress-responsive in mice and translate to human
tissue. We identifedO-linked-N-acetylglucosamine (O-GlcNAc)
transferase (OGT), an X-linked gene important in regulating
proteins involved in chroma-tin remodeling, as fitting these
criteria. Levels of both OGT and itsbiochemical mark,
O-GlcNAcylation, were significantly lower inmales and further
reduced by prenatal stress. Examination of humanplacental tissue
found similar patterns related to X chromosomedosage. As a
demonstration of the importance of placental OGT
inneurodevelopment, we found that hypothalamic gene expressionand
the broad epigeneticmicroRNAenvironment in theneonatal brainof
placental-specific hemizygous OGT mice was substantially
altered.These studies identified OGT as a promising placental
biomarker ofmaternal stress exposure that may relate to sex-biased
outcomes inneurodevelopment.
O-glycosylation | extra-embryonic tissue | neuropsychiatric
disorders
Maternal stress has been identified as a key risk factor
forneurodevelopmental disorders, including schizophrenia andautism,
which often exhibit a sex bias in rates of presentation, ageof
onset, and symptom severity (1–4). Specifically, males exposedto
stress during the first trimester have an increased risk
forschizophrenia, suggesting early pregnancy may be a
sensitivewindow of developmental vulnerability (5). Such
epidemiologicalfindings also support a sex specificity of effects
during a period ofrapid fetal and placental development. Further,
sex differencesin gene expression in the placenta may represent
unique modesof increased disease risk or resilience (4,
6).Alterations in prenatal programming associated with neuro-
developmental disorders likely involve complex interactions
be-tween the maternal environment, the placenta, and factors of
thedeveloping fetus, including sex (4, 6). In eutharian mammals,
in-cluding humans and mice, the placenta serves to mediate
commu-nication between the maternal and fetal compartments,
deliveringnutrients and oxygen and protecting the developing fetus
fromenvironmental insults (7). Although derived from both
maternaland fetal contributions, the majority of the placenta
develops outof the trophoblastic lineage of fetal origin, yielding
a predominantlyXX or XY placenta, from which sex differences in
response tootherwise similar intrauterine perturbations may emerge
(8, 9).In our mouse model of early prenatal stress (EPS), we
have
previously established that male offspring exposed to
maternalstress during early gestation exhibit endophenotypes
associatedwith neurodevelopmental disease, including stress
dysregulationand cognitive deficits that persist into the second
generation,confirming early pregnancy as a point of increased
susceptibilityto the effects of maternal stress experience (5,
10–12). Dynamic
growth and programming of the placenta occurs during this
earlyperiod, and therefore alterations in its function resulting
frommaternal stress exposure could ultimately affect
neurodevelopment(13–16). Therefore, we have used a genomic and
proteomics ap-proach to identify potential placental biomarkers
predictive ofprenatal stress exposure. Our proposed criteria for
biomarkersassumed that candidate genes would (i) show a significant
sexdifference in expression, (ii) be significantly altered in our
EPSmodel, and (iii) be similarly regulated in human placental
tissue.These criteria were established based upon the hypothesis
that asex bias in disease risk stems from basal sex differences in
abilitiesto adapt or respond to a perturbed environment.
ResultsEffects of Offspring Sex and EPS on Placental Gene
Expression. Toidentify placental genes with sex differences in
patterns of expres-sion consistent across gestation [embryonic day
(E) 12.5, E15.5,and E18.5], we used a genome-wide microarray
analysis. Thesegestational time points were selected because they
reflect maturingstages of fetal development, representing likely
important changesin placental function and interactions with the
maternal environ-ment (17). Therefore, we hypothesized that
identification of geneswith sex differences in expression
consistent across this periodwould indicate important candidates
potentially responsible forsex differences in programming outcomes
in response to a per-turbed maternal environment. Differential
expression analyses [falsediscovery rate (FDR) < 0.05] revealed
dynamic developmentalchanges in gene expression patterns, with
2,658 placental geneschanging expression from E12.5 to E18.5.
Surprisingly, sex differ-ences were only found for 11 X- and
Y-linked genes, of which 4 areX and Y paralogs (Fig. 1 A and B and
Table S1). Eight of thesegenes exhibited significant sex
differences in patterns of expres-sion across pregnancy (Table S1).
As expected, X-linked geneswere higher in females, and Y-linked
genes were higher in males.To determine any effects of EPS on
sexually dimorphic genes,
messenger RNA (mRNA) was compared between male andfemale control
or EPS placentas (Fig. 1C). One gene, O-linked-N-acetylglucosamine
transferase (OGT), an X-linked gene with lowerbaseline expression
in male placentas, also had a reduction of ex-pression with the
administration of EPS (Fig. 1D and Dataset S1). Itshould be noted
that EPS had no effect on litter size or sex ratiosin this study
(Dataset S1), as was previously reported (10).
Characterization of Placental OGT Protein, Enzymatic Activity,
andChromatin State at the Ogt Locus. To determine any chromatin
reg-ulation associated with the locus, we measured histone H3
tri-methyl Lys4 (H3K4me3), a permissive chromatin mark, at
thepromoter region (Fig. 1E). Consistent with the basal sex
differences
Author contributions: C.L.H. and T.L.B. designed research;
C.L.H., C.P.M., and D.B.F. per-formed research; C.L.H. and C.P.M.
analyzed data; and C.L.H. and T.L.B. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.1To whom correspondence
should be addressed. E-mail: [email protected].
This article contains supporting information online at
www.pnas.org/lookup/suppl/doi:10.1073/pnas.1300065110/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1300065110 PNAS | March 26,
2013 | vol. 110 | no. 13 | 5169–5174
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A BF12vsM12 F15vsM15
F18vsM18
0
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0
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0 0.5 1.0 1.5 2.0Relative Expression
F/CF/EPSM/CM/EPS
XistKdm5c
Eif2s3xPramel3
Taf1
Rpsa
Ogt
Ddx3yUty
Eif2s3y
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*
*
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*
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% o
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NA
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Fig. 1. Identification of OGT as a potential biomarker of
maternal stress. (A) Venn diagram of developmental differential
expression analyses of mouseplacental genes via Affymetrix GeneChip
Mouse Gene 1.0 ST microarray. The labels of each circle refer to
specific embryonic time point comparisons rep-resented: E12vsE15
are the comparisons between E12.5 (n = 6) and E15.5 (n = 6),
E15vsE18 are the comparisons between E15.5 and E18.5 (n = 6), and
E12vsE18are the comparisons between E12.5 and E18.5. The numbers
within the diagram represent the number of genes characterized as
having differential expressionbetween these groups, independent of
sex, using a false discovery rate of 0.05. (B) Venn diagram of sex
differences in mRNA expression levels analyses ofmouse placental
genes via microarray. Circle labels refer to the sex comparisons
made during each developmental time point. The numbers are derived
as inA. (C) Genes identified as having sex differences in
expression examined in our EPS model. C, control; F, female; M,
male (n = 7); EPS, early prenatal stress (n =8). Data for Xistwere
normalized to the female control levels; data for all other traits
were normalized to male control levels. Bars are the maximum
likelihoodestimate for each group ± the 95% confidence interval for
that estimate. Symbols (* for sex and # for EPS) indicate a main
effect with a confidence intervalthat does not bound zero as
determined by the linear model (y ∼ Sex + EPS + Sex*EPS). (D) OGT
was identified as fitting our biomarker criteria for havingboth sex
differences in expression and responding to EPS. Symbols (* for sex
and # for EPS) indicate a main effect with a confidence interval
that does notbound zero as determined by the linear model (y ∼ Sex
+ EPS + Sex*EPS). Normalization is as in C. (E) Diagram of the
promoter region of OGT used for ChIPanalysis for presence at
H3K4me3. (F) ChIP analysis of the OGT promoter demonstrating the
expression changes in OGT are associated with the presence
orabsence of the transcriptional activator mark, H3K4me3. Data are
representative of the amount of DNA (normalized to the amount of
input ornonimmunoprecipitated DNA) from the promoter region of OGT
associated with the H3K4me3 chromatin mark. Bars are the maximum
likelihood estimatefor each group ± the 95% confidence interval for
that estimate. Asterisk indicates a measurable difference between
female control and all other groups asdetermined by nonoverlapping
confidence intervals for these estimates.
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and EPS pattern of expression found for OGT, we detected a
de-creased association of this locus with H3K4me3 in both male
andin EPS placentas by chromatin immunoprecipitation (ChIP) (Fig.1F
and Dataset S1).Changes in OGT protein levels corresponded with
those found
for mRNA, with less protein in males compared with females,and
less protein in male EPS placentas compared with controlmales (Fig.
2 A and B and Dataset S1). We compared total levelsof O-GlcNAc
modified proteins as a biochemical readout of OGTenzymatic
activity. We observed robust decreases of this mark inmale
placentas compared with females (Fig. 2C and Dataset S1)but no
overall difference in O-GlcNAcylation between male con-trol or EPS
placentas (Dataset S1). Despite no overall difference,two bands at
28 and 37 kDa, which were visibly different betweenmale control and
EPS placentas, were excised for proteomicanalysis (Fig. 2D). These
proteins were identified as annexin A1(ANXA1) and peroxiredoxin 1
(PRDX1) (25 and 21, respectively,unique peptide sequences
identified with >95% probability forthese proteins; Fig. 2 E and
F and Table S2). Total protein levels ofboth ANXA1 and PRDX1 were
not affected by EPS (Fig. S1 A andB and Dataset S1). There were no
global sex differences in levelsof total serine or threonine
phosphorylation, demonstrating thatall posttranslational
modifications were not affected in the samemanner as
O-GlcNAcylation (Fig. S2 A and B and Dataset S1).
Effect of X Chromosome Complement on Human Placental OGT mRNAand
Protein. To determine the translational potential of our find-ings,
OGT levels were evaluated in human term placenta. Biopsieswere
obtained from male placentas, enriched for fetal (XY) ormaternal
(XX) contributions to assess X-chromosomal comple-ment effects. OGT
was highly expressed in human placenta fromboth maternal and fetal
contributions. Similar to our findings in
mice, gene expression in XY samples was measurably lower forOGT
compared with XX samples (Fig. 2G and Dataset S1).Biochemical
analysis of O-GlcNAc modified proteins followedthis same pattern
(Fig. 2H and Dataset S1).
Reduced Placental OGT Results in Broad Neurodevelopmental
Changes.To determine the potential programming effects that
reducedplacental OGT would impose on the developing brain, we used
aplacental-specific Cre-recombinase–expressing mouse to
condi-tionally target the Ogt gene (18). Using this genetic model,
femalemice had reduced placental OGT expression but no differencein
the liver (Dataset S1). We examined broad patterns of
geneexpression in the neonatal hypothalamus, because we weremost
interested in changes that would potentially affect neu-roendocrine
systems. In addition, we compared neonatal whole-brain microRNA
patterns as a marker of the potential differencesin the epigenetic
environment in the developing brain. BecauseOGT is X-linked,
wild-type and hemizygous females were com-pared because our goal
was to examine a model of controlledreduced OGT, and not a knockout
of this gene, which would bethe result in males. We detected robust
changes in hypothalamicgene regulation, with 370 genes showing
significant differences(FDR < 0.05; Fig. 3A and Dataset S2).
Further, functional anno-tation clustering of these affected genes
revealed a significantenrichment for pathways involved in energy
utilization, proteinregulation, and synapse formation, processes
important in neuro-development (Fig. 3A) (19, 20). Similar to the
extensive gene ex-pression changes we detected in the hypothalamus,
we also foundrobust differences in the brain microRNA environment,
wherehemizygous placental OGT expression shifted the pattern of
250of the most abundant brain microRNAs to be distinct from that
ofwild-type females by hierarchical clustering analyses (Fig.
3B).
A B
C D
E
F
G H
Fig. 2. Biochemical assessment of OGT and O-GlcNAcylationin
mouse and human placentas. (A) Representative Westernblot images of
OGT levels from male and female mouse pla-centas. Histogram is the
maximum likelihood estimate for thenormalized optical densities of
each group (n = 7) ± the 95%confidence interval for that estimate.
Asterisk indicatesmeasurable difference between groups as
determined bynonoverlapping confidence intervals for the estimates.
(B)Representative Western blot images of OGT levels fromcontrol (n
= 7) and EPS (n = 8) male mouse placentas. Histo-gram was derived
and annotated as in A. (C ) RepresentativeWestern blot images of
total O-GlcNAcylated proteins frommale and female mouse placentas.
Histogram was derived andannotated as in A (n = 7). (D)
Representative Western blotimages of total O-GlcNAcylated proteins
from male control (n =7) and EPS (n = 8) placentas. The image is
annotated to high-light the bands visibly identified with
differential O-GlcNA-cylation between treatment groups excised for
proteomicanalyses. (E ) Amino acid sequence coverage of
LC-MS/MSwhere the band at 28 kDA was identified as
peroxiredoxin-1(PRDX1) and the band at 37 kDA was identified as
annexin A1(ANXA1) via LC-MS/MS with greater than 99.9% certainty.
(F)Representative spectrum image of peptide fragments used
toidentify both ANXA1 and PRDX1. (G) Expression of OGT by RT-PCR in
human placental tissue associated with male births. Datawere
normalized to XY levels. Bars are the maximum likeli-hood estimate
for each group (n = 4) ± the 95% confidenceinterval for that
estimate. XX, maternal; XY, fetal. (H) Rep-resentative Western blot
images of total O-GlcNAcylatedproteins from both XX and XY
placental contributions fromhuman placentas. Histogram was derived
as in A (n = 4). As-terisk indicates a measurable difference
between groups withdifferent X-chromosome complement as determined
by non-overlapping confidence intervals for each estimate.
Howerton et al. PNAS | March 26, 2013 | vol. 110 | no. 13 |
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DiscussionAn increased risk for neurodevelopmental disorders
such asschizophrenia has been associated with fetal antecedents
in-cluding prenatal stress. Because such diseases often present
witha large sex bias in rates of diagnosis, age of onset, and
symptomseverity, we used a genomics approach to identify
potentialplacental candidate genes predictive of prenatal stress
exposurefrom male and female placental tissue taken at E12.5,
E15.5, andE18.5 (1–4). From this screen, OGT was identified as an
importantplacental effector of programmatic changes in
neurodevelopment,providing a potential mechanism for
neurodevelopmental changesresulting from maternal stress in early
pregnancy (EPS). The cri-teria used in this assessment included
that the candidate gene must(i) show a significant sex difference
in expression across preg-nancy, (ii) be significantly altered in
our EPS model, and (iii) besimilarly regulated in human placental
tissue. The hypothesis inthese studies centered on a potential
threshold effect wherebybasal sex differences in placental gene
expression may provide asex bias for risk or resiliency in response
to prenatal insults.OGT is a key cellular regulator through the
unique post-
translational modification it places on serine and threonine
res-idues of both nuclear and cytosolic proteins. More
specifically,OGT plays an important role in programmatic chromatin
re-modeling by O-glycosylation (O-GlcNAcylation) of protein
tar-gets, including RNA polymerase II, histone deacetylases,
andhistone 2B (21). Because O-GlcNAcylation competes with
ser-ine/threonine phosphorylation, it serves a critical function in
theregulation of enzymatic activity important in somatic cell
func-tion and embryo viability (22–25). Additionally, OGT has
beencharacterized as a cellular nutrient sensor (26) and therefore
mayplay an important role in the protective effects of the placenta
onthe developing brain from insults such as maternal food
depri-vation (7). We hypothesized that levels of this important
enzymebelow a critical point following maternal stress could
significantlyaffect specific aspects of placental function.
Therefore, becausemale placentas showed a lower baseline expression
of OGT, astress-mediated further reduction may specifically place
malefetuses at a disadvantage in being able to adapt to a
changingenvironment, and at an increased risk for long-term effects
on
neurodevelopment. Additionally, OGT has been characterizedas a
cellular nutrient sensor (26).Levels of OGT mRNA, protein, and
O-GlcNAc were all re-
lated to X-chromosome complement, suggesting this gene
likelyescapes X inactivation in the placenta. These levels were
allfurther reduced in our mouse model of EPS. Factors modulatingOGT
expression and translation have not been well character-ized.
However, biallelic expression in female embryonic stemcells at
varying stages of differentiation has been reported (27,28). As
further evidence for regulation of OGT expression at
atranscriptional level, ChIP was conducted at the Ogt locus
withH3K4me3, a permissive chromatin mark indicative of
transcrip-tional activation. A pattern of association with this
activationalmark was found similar to that detected for OGT mRNA
andprotein levels, where there was a reduction for males and
EPSgroups compared with control females. Interestingly, the
his-tone demethylases, lysine-specific demethylase 5C (KDM5C)and
lysine-specific demethylase 5D (KDM5D), which demethylateH3K4me3,
were genes also identified as having a sex-dependentexpression
pattern in our placental array. Taken together, thesedata support
additional modes of sex-dependent epigenetic reg-ulation whereby a
potential imbalance in chromatin regulationmay affect placental
function.In addition to OGT identification in this genomic screen
as an
important candidate placental biomarker, additional genes
ofinterest were also noted. Overall, there were dynamic changes
ingene expression patterns and within unique gene sets over
thethree gestational time points examined. Despite the
dynamicnature of this tissue, only a small subset of genes
demonstratedsex differences in patterns of expression, all of which
were sexchromosome-linked: Ogt; X-inactive specific transcript
(Xist);Kdm5c; eukaryotic translation initiation factor 2, subunit
3,structural gene X-linked (Eif2s3x); preferentially expressed
anti-gen in melanoma-like 3 (Pramel3); TATA box binding
protein-associated factor 1 (Taf1); ribosomal protein SA (Rpsa);
DEADbox polypeptide 3, Y-linked (Ddx3y); ubiquitously
transcribedtetratricopeptide repeat containing, Y-linked (Uty);
eukaryotictranslation initiation factor 2, subunit 3, structural
gene Y-linked(Eif2s3y); and Kdm5d. These genes have functions
involved inprotein translation, histone methylation, and protein
glycosylation
A B
NADH ProcessingRibosomal FunctioningElectron Transport Chain
Mitochondrial Structure
Nuclear Envelope
Functional groups downregulatedwith reduced placental Ogt
UbiquitinationReceptor SignallingSynaptic Plasticity
WD40 Domain Proteins
Proteolysis
Functional groups upregulatedwith reduced placental Ogt
F P.Cre -F P.Cre + F P.Cre -
F P.Cre +
Fig. 3. Placental specific reduction of OGT broadly affectsearly
neurodevelopment. (A) Heat map of PN2 hypothalamicgenes with
significantly different levels of expression be-tween XOgt/ XWT
females, with or without placental Creexpression [P.Cre+ (n = 4) or
P.Cre– (n = 5), respectively],resulting in a 50% reduction in OGT
expression in the P.Cre+females (Fig. S3). Genes that were either
significantly up-regulated (n = 218) or significantly
down-regulated (n = 161)with reduced placental OGT, as determined
by a FDR of 0.01,were grouped into functional annotation categories
using theDAVID functional annotation clustering tool. (B) Heat map
ofwhole-brain microRNA expression levels from the same ani-mals in
A. Hierarchical clustering discriminates all but onesample between
the two groups [P.Cre+/− (n = 5) vs. P.Cre−/−
(n = 4)], suggesting broad epigenetic changes in the PN2
brain.
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(Table S1). Although these genes showed significant sex
differ-ences, no further effect of EPS was detected. However, the
sexdifferences in their patterns of expression suggest that they
mayplay an important role in sex differences in placental function
thatcould also contribute to a sex bias in neurodevelopmental
disease.Further, the surprisingly limited number of genes
exhibiting sexdifferences in expression in the placenta across
pregnancy usingour strict statistical requirements highlights the
unique control ofgene expression in the placenta and the potential
impact pertur-bations during gestation may have on sex biases in
programmingoutcomes in the developing fetus.In the biochemical
assessment of OGT activity, O-GlcNAc
levels were significantly reduced in male placentas, fitting
withtheir decreased OGT mRNA and protein. In response to EPS,we
noted two distinct bands in male placental samples that
wereapparently reduced. These proteins were identified as ANXA1and
PRDX1 by proteomics analyses. Both proteins are knowntargets of
OGT; however, the exact site of O-glycosylation hasnot been
determined (29). ANXA1 is an important modulator ofcellular
inflammatory signaling, and PRDX1 is an intracellularperoxidase
that functions as a chaperone with clients includingnuclear
factor-kappa β (30–34). These are two specific examplesof
mechanisms by which EPS may produce sex differences in pla-cental
function that could affect brain development trajectories.To
determine the translational potential of our findings, OGT
levels and biochemistry were evaluated in human term
placentaltissue. Biopsies obtained from male placentas enriched for
fetal(XY) or maternal (XX) contributions to assess
X-chromosomalcomplement found that OGT was highly expressed and,
similarto our findings in mice, XY samples were significantly lower
forOGT compared with XX samples. Biochemical analysis of
O-GlcNAcylated proteins followed this same pattern (Fig. 2H).Human
placental tissue was obtained from deidentified cesarean-section
deliveries, and thus the pregnancy experience and exposureto
various stressors or other fetal antecedents could not
bedetermined. As such, to avoid likely confounding of these
un-controllable variables, only X-chromosomal dosage effects onOGT
expression were evaluated (i.e., frommale births). Together,these
data support OGT as meeting all three of our criteria asa
predictive placental biomarker.To examine a more direct link
between reduced placental OGT
and potential programming changes in the developing brain,
weused a placental-specific Cre-recombinase–expressing mouse
toconditionally target the Ogt gene. We examined broad patternsof
gene expression in the neonatal hypothalamus, because wewere
interested in programming changes that would potentiallyaffect
neuroendocrine systems based on our EPS model, byAffymetrix
microarray (12, 35). In addition, we compared neo-natal whole-brain
microRNA patterns by ABI Taqman array asa marker of the potential
differences in the epigenetic environ-ment in the developing brain,
and based on our previous findingsfor dynamic microRNA changes in
our EPS model (35). BecauseOGT is X-linked, wild-type and
hemizygous females were com-pared to examine a model of controlled
reduced OGT, and not aknockout of this gene, which would be the
result in males. Im-portantly, gene expression changes as a result
of reduced pla-cental OGT in this context are representative of the
impact thisbiomarker has on neurodevelopment irrespective of
chromo-somal sex and were not directly compared with the effects of
EPSon early brain development. We detected profound changes
inhypothalamic gene regulation, with over 375 genes showing
sig-nificant differences in expression patterns in the
hypothalamusfrom placental hemizygous Ogt mice compared with
controls.Further, functional annotation clustering of these
affected genesrevealed a significant enrichment for pathways
involved in energyutilization, protein regulation, and synapse
formation, all pro-cesses important in neurodevelopment (19, 20).
Similar to theextensive gene expression changes we detected in the
hypothal-amus, we also found robust differences in the brain
microRNAenvironment, where hemizygous placental OGT expression
shiftedthe pattern of 250 of the most abundant brain microRNAs to
be
distinct from that of control animals by hierarchical
clusteringanalyses. These results support our previous studies
showing similarbroad changes produced by EPS in the postnatal brain
microRNAenvironment indicative of epigenetic programming
involvement inshaping plasticity during neonatal brain development
(35). Thesedata substantiate the importance of regulation of
placental OGTlevels and provide a potential link with profound
changes in genetranscription in neurodevelopment with a candidate
biomarker.The endocrine placenta is poised to be a key mediating
tissue
in responding to a dynamic and changing maternal environment.Sex
differences in how this tissue responds to the same maternalmilieu
likely contribute to the altered susceptibility of male andfemale
fetuses to long-term programming outcomes (4). Ourstudies
identified OGT, an intracellular glycotransferase impor-tant in
regulating key chromatin programming events, as a po-tential
placental biomarker that was sex-biased in its
expression,responsive to EPS, and similar in X-linked expression
pattern inhuman tissue. Because the placenta is readily accessible,
thesestudies have high translational potential and may be useful
inpredicting gestational stress exposure.
MethodsAnimals. Male C57BL/6J and female 129S1/SvImJ mice were
obtained fromJackson Laboratories and subsequently used as breeding
stock to produceC57BL/6J:129S1/SvImJ hybrids (F1 hybrids). F1
hybrid breeding pairs (n = 33)were checked daily at 7:00 AM for
copulation plugs. Noon on the day thatthe plug was observed was
considered to be embryonic day 0.5 (E0.5). F1hybrids were used for
the placental microarray and the prenatal stressexperiments. For
the placental-specific reduction of OGT, double heterozy-gous
[B6.129-Ogttm1Gwh/J (XOgt/ XWT); B6-CYP19-Cre (placental-Cre
recombi-nase heterozygote, P.Cre+/−)] females (n = 17) were bred to
hemizygousB6.129-Ogttm1Gwh/J (XOgt/ Y)/ heterozygous (P.Cre+/−)
males (n = 14),resulting in offspring representing all potential
genotypes. To identify theeffects of a placental-specific reduction
of OGT, female XOgt/ XWT P.Cre+/−
(P.Cre+/−, n = 10) were compared with XOgt/ XWT P.Cre−/−
(P.Cre+/−, n = 10)for placental specificity validation and brain
analyses. All animal protocolswere reviewed and approved by the
Institutional Animal Care and Use Com-mittee of the University of
Pennsylvania.
Early Prenatal Stress. Administration of chronic variable stress
was performedas previously described (12). Dams were randomly
assigned to treatmentgroups to receive stress during days 1–7 of
gestation (EPS; n = 8) or to acontrol (n = 7) nonstressed group.
Pregnant mice assigned to the EPS groupexperienced each of the
following stressors on a different day of the EPSperiod: 60 min
(beginning at 1:00 PM) of fox odor exposure (1:5,000
2,4,5-trimethylthiazole; Acros Organics), 15 min of restraint
(beginning at 1:00 PM)in a modified 50-mL conical tube, 36 h of
constant light, novel noise (WhiteNoise/Nature Sound-Sleep Machine;
Brookstone) overnight, three cagechanges (at ∼9:00 AM, 12:00 PM,
and 3:00 PM) throughout the light cycle,saturated bedding (700 mL,
23 °C water) overnight, and novel object (eightmarbles of similar
shape and color) exposure overnight. These stressors wereselected
to be nonhabituating and did not induce pain or directly
influencematernal food intake, weight gain, or behavior (10).
Mouse Tissue Dissection. For the placental microarray experiment
mouseplacentas and embryonic somatic tissue were dissected at
E12.5, E15.5, andE18.5 (n =6 at each gestational time point). DNA
was extracted from theembryonic somatic tissue, and the sex of the
embryo was determined usingmethods described elsewhere (36). One
male and one female placenta fromeach litter were used for
microarray analysis; these placentas were quarteredand put in 500
μL of RNAlater and stored at −80C until processed as below.For the
prenatal stress experiment, E12.5 male and female placentas
fromcontrol (n = 7) and EPS (n = 8) litters were used for gene
expression, ChIP,and protein analyses; these placentas were
bisected via transverse sectioningand one half was placed in 500 μL
of RNAlater and stored at −80C (for geneexpression analyses) and
one half was flash-frozen in liquid nitrogen andstored at −80C (for
ChIP and Western blot analyses).
For the placental-specific reduction of OGT experiment, two
cohorts ofanimals were used: (i) an embryonic cohort (n = 4
P.Cre+/− and P.Cre−/−) tovalidate the specificity of OGT reduction
to the placenta and (ii) a cohort (n = 4P.Cre+/−; n = 5 P.Cre−/−)
killed on postnatal day 2 (PN2) for brain analyses. Forthe
validation, placentas and embryonic somatic tissue were dissected
at E12.5.DNA was extracted as above; placentas and liver tissue
were flash-frozen in
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liquid nitrogen and stored at −80C. For the brain analyses, pups
were killedat PN2 and brains were dissected and flash-frozen in
liquid nitrogen andstored at −80C. Tissue punches (300 μm) of the
hypothalamus [∼3.0–3.3 mmposterior in the anterior–posterior plane
and 1.5 mm ventral in the dorsal–ventral plane, structures
corresponding to the paraventricular area in a de-velopmental mouse
brain atlas (37)] were collected using a 1.0-mm circularpunch (Ted
Pella) and then used for microarray analysis; the remaining
braintissue was used for microRNA analysis.
Human Tissue Samples. Placental tissues associated with
deidentified malefetuses (n = 4) were quartered by cutting in the
transverse section throughthe location of the emergence of the
umbilical cord in two directions. Bi-opsies (∼5 mg) were obtained
from both the fetal and maternal con-tributions to the placenta
from three locations, 5 cm from the umbilical cord.Biopsies were
flash-frozen in liquid nitrogen and stored at −80 C until fur-ther
analysis. Human tissue collection protocols were reviewed and
ap-proved by the Institutional Board of the University of
Pennsylvania.
Brain microRNA Environment. Total RNA was extracted from brains
[P.Cre+/−
(n = 4) and P.Cre−/− (n = 5)] using TRIzol reagent (Invitrogen).
One micro-gram of total RNA was reverse-transcribed to cDNA using
Megaplex RT poolA primers and Multiscribe reverse transcriptase
(Applied Biosystems). Ex-pression levels of 245 microRNAs were
determined using the Taqman ArrayMicroRNA card A Array (Applied
Biosystems). Analysis was performed usingthe comparative cycle
threshold (Ct) method. For each sample, the averageof the Ct values
of small nucleolar RNA (sno)135 and sno202 was used as anendogenous
loading control. Expression levels of each sample were
normalized
to the average expression level of P.Cre−/− females. Uninformed
hierarchicalclustering using Pearson correlations was used to
discriminate differentialmicroRNA expression between individuals
(42).
Statistical Analyses. Analyses were performed using R (version
2.14.2) and thepackages arm, (43), bbmle (44), and limma (39, 45)
to fit the gene expressionand optical density data to linear
models, and estimates for main and inter-action effects were
determined from these models. For all analyses, maximumlikelihood
estimates were calculated and 95% confidence intervals
wereconstructed for regression coefficients as the reported
statistical values.We used this statistical approach over the more
traditional null hypothesissignificance testing to provide the
magnitude for any observed experi-mental effect, to provide a
measure of dispersion around this effect sizeto allow assessment of
the predictive value of our models, and to relateany statistical
conclusions to the experimental hypothesis (46). A confi-dence
interval for an estimate that did not bound zero was considered
tobe significant; comparisons among experimental groups were made
usingconfidence interval evaluation, and those that did not overlap
were con-sidered to be significantly different. Database for
Annotation, Visualizationand Integrated Discovery (DAVID)
functional annotation clustering (47)was used for the PN2
hypothalamic analysis, and MeV v4.8 was used forthe PN2 microRNA
analysis.
ACKNOWLEDGMENTS. We thank C. Taylor for technical assistance
andanimal care and G. Leone at The Ohio State University for the
generousdonation of Cyp19-Cre mice. This work is supported by
National Institutes ofHealth Grants MH099910, MH091258, and
MH087597.
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