the concept of the fetal genome is no longer that of a static framework inherited from paternal 
 and maternal sources but a malleable scaffold constantly adapting to stimuli .
this is most evident 
 in studies involving fetal programming due to the effects of nutritional variation and 
 glucocorticoid exposure . here , we examined the resulting 
 fetal molecular preconditioning due to antenatal steroid therapy using a protein - restriction mouse 
 model .
this model was first designed in a previous study 
 as a novel approach to evaluate postnatal adaptive responses due to varied prenatal nutritional 
 conditions and the addition of stress or steroids .
molecular evidence revealed that prenatal 
 programming secondary to maternal protein restriction rendered an inherent susceptibility to neural 
 compromise in neonates and any further addition of antenatal steroids may be detrimental to these 
 already injury - prone offspring .
thus , an examination of underlying molecular mechanisms in the fetus 
 was warranted to elucidate the effects seen postnatally .
understanding any subtle changes in the fetus induced by these factors and their correlation with 
 phenotypic outcomes in the adult would facilitate early detection of either well - being or disease . 

current biomolecular techniques such as microarray analysis have allowed the investigation of global 
 gene expression and subsequently , the parallel data mining of gene transcripts of interest as well 
 as the discovery of new gene involvement .
moreover , gene expression profiles through clustering of 
 significant genes have shown promising potential as diagnostic panels .
all these have led to the 
 rapid identification of biomarkers for disease conditions and their associated regulatory pathways 
 . using these advancements , a panoramic view of genetic 
 movement in utero
female c57bl/6n mice about 6 weeks old provided by the institute for animal experimentation , 
 tohoku university graduate school of medicine , were maintained under controlled lighting ( 12-hour 
 light cycles ) and temperature ( 24c ) .
these were allowed for free access to food ( ain-93 g : 
 oriental yeast co. , ltd . , tokyo , japan ) and water during a 2-week acclimatization period after which 
 each female was time mated with a male .
pregnant females ( n = 36 ) were then housed singly and administered either 
 control ( c ) or protein restricted ( pr ) diets ad libitum all throughout pregnancy ( embryonic stage , 
 e0 to e17 ) .
these were then further subdivided into 6 groups and subjected to either plain 
 normosaline solution ( c - s , pr - s ) or 100  g / kg dexamethasone sodium 
 phosphate ( decadron , msd k.k . ,
tokyo , japan ) in normosaline solution ( c - d / s , pr - d / s ) by subcutaneous 
 injection daily during late gestation ( e10 to e17 ) .
maternal weights on days e0 , 
 e10 , and e17 were recorded , as well as fetal weights on e17 . on embryonic day 17
, whole brain 
 samples collected from 2 male and 2 female fetuses from each litter were supercooled in liquid 
 nitrogen and stored at 80c .
a total of 6 toray 3d - gene mouse oligo chip 24  k ( toray industries , inc . , tokyo , japan ) 
 microarrays were analyzed per treatment .
each chip utilized a 
 0.5  g portion of combined total rna from a matched pair of male 
 and female samples .
rna was amplified and labeled using an amino allyl messageamp ii arna 
 amplification kit ( life technologies japan ltd . ) according to the manufacturer 's instructions .
each 
 sample of arna was labeled with fluorescence cy3 or cy5 and cohybridized at 37c for 16 
 hours .
hybridization signals were scanned using scan array 
 express ( perkin elmer , ma , usa ) and global background analysis was performed using genepx pro ( mds 
 analytical technologies , ca , usa ) .
all 36 arrays were then normalized together as one experiment to 
 reduce nonbiological variability . to validate microarray results ,
qpcr was performed on 2 selected genes , microtubule - associated 
 protein 1b ( mtap1b ) and 3-hydroxy-3-methylglutaryl - coa synthase 1 
 ( hmgcs1 ) , using the c and c - d / s treatments .
total rna was extracted from whole 
 fetal brains ( n = 6 per treatment ) using qiazol lysis reagent ( qiagen , hilden , 
 germany ) and cleaned with an allprep dna / rna mini kit ( qiagen , hilden , germany ) according to the 
 manufacturer 's protocol .
complimentary dna was synthesized using the superscript iii first - strand 
 synthesis system ( invitrogen , carlsbad , ca ) and quantitative pcr was conducted with express sybr 
 greener supermix with premixed rox ( invitrogen , carlsbad , ca ) on an eppendorf 
 realplex mastercycler ( eppendorf , hamburg , germany ) . amplified 
 transcripts were quantified and normalized against hypoxanthine phosphoribosyltransferase 1 
 ( hprt1 ) .
microarray data were subjected to t - test analyses with standard bonferroni 
 correction for multiple comparisons .
the p value was set at 0.05 and a threshold of 
 1.5-fold was applied to determine significantly regulated genes .
these were subjected to an 
 ontological review and a subset of neurodevelopmental genes for genetic profiling was determined by 
 hierarchical clustering .
targeted gene transcripts of interest on the microarrays were treated to 
 one - way anova with post - hoc analysis ( bonferroni post - test ) .
confirmation of selected genes by qpcr 
 was validated through fold change analysis ( threshold of 1.5-fold ) .
data management , statistical 
 analysis , and gene ontology were performed using geworkbench software ( https://gforge.nci.nih.gov/frs/?group_id=78 ) and mgi gene ontology tools ( http://www.informatics.jax.org/gotools/ ) .
mean maternal weight gain patterns , between groups , were similar before ( e0 to e10 ) and during 
 treatment ( e10 to e17 ) .
mean fetal brain to body weight indices on sampling day e17 were not 
 significantly different ( figure 1 ) .
microarray analyses of 23,522 probe transcripts presented 10,946 genes without absent calls or 
 unreadable hybridization signals .
there were more upregulated genes as compared to downregulated 
 genes across all treatment groups ( figure 2 , table 1 ) .
the combined number of significant genes regulated from 
 all treatment groups versus the control was 332 ( figure 3 ) . 

subsequent gene ontology analysis revealed that ongoing cell organization and biogenesis , 
 developmental processes , and transport were most rampant in the global expression survey .
the 
 discovery of genes uniquely activated per treatment and sharing similar ontologies facilitated 
 individual treatment characterization ( table 2(a ) ) . 

associated genes were found for protein restriction , cell adhesion genes in both pr - d / s only 
 ( col1a1 , atp1b2 , ctnnd1 , rpsa , 
 and fat4 ) , and pr - s only ( cdh2 , edil3 , 
 and astn1 ) ; for dexamethasone treatment , stress response genes in both c - d / s only 
 ( brsk1 , rarres2 ) , and pr - d / s only ( klk8 , 
 myo6 , ndufa6 , col1a1 , mapk8 , and 
 phlda3 ) ; and for both protein restriction , dexamethasone treatment , and dna metabolism 
 genes for pr - d / s only ( tcf3 , mapk8 ) .
overall , neurodevelopmental 
 genes were overrepresented among those significantly regulated ( table 2(b ) ) and were associated with nervous system development 
 ( pbx3 , eif2b5 , nlgn1 , 
 mark4 , atp2b2 , nrxn3 , ncam1 , 
 tnik , slitrk1 , cdh2 , synj1 , 
 palm , nrp1 , rpl24 , mtap2 , 
 rpgrip1 , pou3f2 , gabrb3 , lrp6 , 
 sulf2 , ank3 , ccdc88a , atrx , 
 nr2c2 , opa1 , abi2 , mtap1b , 
 tcf3 , syne1 , mapk8 , golga2 , 
 atxn2 , gfra1 , snap91 , slitrk5 , 
 celsr2 , emx2 , klk8 , myo6 , 
 scn2a1 , sema3c , and kif5c ) ; generation of 
 neurons ( pbx3 , nlgn1 , atp2b2 , 
 ncam1 , tnik , slitrk1 , cdh2 , 
 synj1 , palm , nrp1 , rpl24 , 
 mtap2 , rpgrip1 , pou3f2 , gabrb3 , 
 lrp6 , ank3 , ccdc88a , abi2 , 
 mtap1b , tcf3 , syne1 , mapk8 , 
 golga2 , atxn2 , gfra1 , slitrk5 , 
 snap91 , celsr2 , emx2 , klk8 , 
 myo6 , sema3c , kif5c , robo2 , 
 arhgef2 , brsk1 , pou3f4 , and 
 sox11 ) ; neuron differentiation ( pbx3 , nlgn1 , 
 atp2b2 , ncam1 , tnik , slitrk1 , cdh2 , palm , nrp1 , rpl24 , mtap2 , rpgrip1 , pou3f2 , gabrb3 , lrp6 , ank3 , 
 ccdc88a , abi2 , mtap1b , tcf3 , syne1 , mapk8 , golga2 , gfra1 , atxn2 , slitrk5 , snap91 , celsr2 , emx2 , 
 klk8 , myo6 , sema3c , kif5c , robo2 , brsk1 , pou3f4 , and sox11 ) ; 
 neurogenesis ( pbx3 , eif2b5 , nlgn1 , atp2b2 , ncam1 , tnik , slitrk1 , cdh2 , synj1 , palm , 
 nrp1 , rpl24 , mtap2 , rpgrip1 , pou3f2 , gabrb3 , lrp6 , ank3 , ccdc88a , abi2 , mtap1b , tcf3 , syne1 , mapk8 , 
 golga2 , atxn2 , gfra1 , slitrk5 , snap91 , celsr2 , emx2 , klk8 , myo6 , sema3c , kif5c , robo2 , arhgef2 , 
 brsk1 , pou3f4 , and sox11 ) ; neuron development 
 ( pbx3 , abi2 , mtap1b , syne1 , mapk8 , golga2 , nlgn1 , atxn2 , gfra1 , snap91 , slitrk5 , celsr2 , 
 atp2b2 , ncam1 , tnik , slitrk1 , klk8 , cdh2 , myo6 , sema3c , palm , kif5c , nrp1 , rpl24 , robo2 , mtap2 , 
 rpgrip1 , brsk1 , pou3f2 , gabrb3 , ank3 , and ccdc88a ) ; and neuron 
 projection development ( abi2 , mtap1b , syne1 , mapk8 , golga2 , nlgn1 , atxn2 , gfra1 , 
 snap91 , slitrk5 , celsr2 , ncam1 , tnik , slitrk1 , klk8 , cdh2 , myo6 , sema3c , palm , kif5c , nrp1 , rpl24 , 
 robo2 , mtap2 , brsk1 , pou3f2 , ank3 , and ccdc88a ) .
a subset of 8 genes out of 332 was filtered through the hierarchical clustering method allowing 
 segregation of treatments ( figure 4 ) .
an assessment of 
 individual biological themes within the subset revealed neurodevelopmental roles and distinct causal 
 relationships with glucocorticoid treatment and protein restriction ( table 3 ) .
the addition of stress or steroids ( -s and -d / s groups ) greatly affected gene regulation leading 
 to further investigation of genes related to glucocorticoid and stress signaling pathways . 

mapk8 , fkbp5 , mkp-1 , pp2a , akt , and gsk3 exhibited expression patterns 
 across treatment groups that corresponded to an overall reduction in glucocorticoid receptor ( gr ) 
 activity in the -d / s and -s groups .
most significant of which were the marked differences between 
 c - d / s and pr - d / s ( mapk8 , gsk3 , and mtap1b ) emphasizing the 
 disparate effect on varying nutritional conditions due to dexamethasone administration ( figures 
 5(a ) and 5(b ) ) .
qpcr results demonstrated a good agreement with the microarray data for mtap1b 
 and hmgcs1 ( figure 6 ) .
the maternal - fetal compartment serves to cushion the fetus from environmental stimuli , but beyond 
 normal circumstances , prenatal conditioning invariably occurs .
microarray analysis allowed for a 
 panoramic view of gene activity along two levels : through the global expression of genes and through 
 individual treatment groups and their association with one another . in general , ongoing cell 
 organization and biogenesis , developmental processes , and transport were most rampant in the global 
 expression survey ( table 2(a ) ) .
the dominance of these 
 particular gene groups is expected in the developing fetus just as genes for growth and maturation 
 are more likely activated in neonates .
regardless , these increased gene frequencies most likely 
 demonstrate consequent fetal reactions to acquired insults from protein restriction and 
 glucocorticoid exposure either as compensatory regulation or protective feedback .
they signaled 
 changes within a fetus long previously believed to be immune prior to the conception of the barker 
 theory .  
this was exemplified by recurring themes in biological processes related to ongoing brain 
 development during fetal stages : multipotent progenitor differentiation and neuronal migration . 

their increased expression over other gene systems emphasized the significance of fetal 
 neuroplasticity even to the detriment of visceral organ growth similar to physiologic brain sparing . 

this also underlined the early dependence on brain - controlled pathways that trigger bodily functions 
 during stages when less developed organs have not yet attained full functional independence .
the isolation of 
 distinct genes associated with individual factors of protein restriction , dexamethasone , and stress 
 were complimentary to known gene networks .
studies of these genes 
 uniquely regulated in both pr - d / s ( col1a1 , atp1b2 , ctnnd1 , rpsa , and 
 fat4 ) and pr - s only ( cdh2 , edil3 , and astn1 ) emphasized 
 the crucial role of nutritional factors in maintaining the integrity of cell interaction . 

col1a1 , a known marker of fibrosis and aging , has been linked to alterations in oxidative 
 and antioxidant defense capacity in cells due to poor maternal nutrition . on the other hand , modifications in atp1b2 , edil3 , 
 and astn1 during development lead to glial dysfunction [ 1416 ] .
moreover , various 
 studies on nutritional factor effects and progenitor cell differentiation included ctnnd1 , 
 fat4 , and cdh2 [ 1719 ] . fetal dexamethasone exposure impairs development in various cell types eliciting a dose - dependent 
 stress response . in the brain ,
the pituitary is the site 
 of action of administered dexamethasone in the blockade of stress induced hypothalamic - pituitary 
 axis ( hpa ) activation .
the latter involves the stimulation of brain receptors , primarily , those of 
 glucocorticoid receptors ( gr ) by both exogenous and endogenous corticosterone . during conditions of 
 stress , the hpa axis releases reactive feedback which suppresses increased excitability allowing 
 recovery from stress induced activation and facilitation of memory storage .
the addition of 
 dexamethasone can partially deplete the brain of corticosterone and in turn suppress the fetal hpa 
 axis .
studies that included the genes uniquely regulated in c - d / s ( brsk1 , 
 rarres2 ) and pr - d / s ( klk8 , myo6 , 
 ndufa6 , col1a1 , mapk8 , and phlda3 ) 
 report their important roles in neuroregulation and adaptation to stress responses during brain 
 development [ 2125 ] .
furthermore , expression patterns of targeted genes related to stress signaling pathways 
 revealed decreased gr activity : mapk8 ( mitogen activated protein kinase 8) 
 and fkbp5 ( fk506 binding protein 5 ) , both gr inhibitors [ 26 , 27 ] , were increased in the -d / s 
 and -s groups ; mkp1 ( mitogen activated protein kinase phosphatase 1 ) and 
 pp2a ( protein phosphatase 2 ) , both map kinase inhibitors [ 28 , 29 ] , were decreased in the -d / s and -s groups 
 ( figure 5(a ) ) .
genes associated with pr - d / s only ( tcf3 and mapk8 ) on 
 microarray analysis , as well as two targeted genes ( akt and gsk3 ) 
 all function in metabolic gene networks , especially for glucose metabolism in dna synthesis . here
, 
 their specific expression patterns between -d / s groups underscored the most significant observation 
 in this study , which was the apparent harmful effect of dexamethasone to fetuses in highly stressed 
 conditions ( pr - d / s ) .
a diagram interrelating the targeted genes of interest with regard to gr 
 activity is shown in figures 7(a ) and 7(b ) .
previous reports have stated that a loss of gr activity reduces 
 dexamethasone inhibition of akt ( thymoma viral protooncogene 1 ) , which in turn 
 decreases akt inhibition of gsk3 ( glycogen synthase kinase 3 ) , a 
 proapoptotic gene [ 3032 ] .
but in the pr - d / s group , a highly 
 stressed condition , dexamethasone was evidently harmful .
this disparate pattern between c - d / s and 
 pr - d / s , significant in mapk8 , gsk3 , and mtap1b ( p 
 <
0.0001 ) , denotes the possibility of ongoing altered neurodevelopment or even 
 neurodysgenesis ( figure 5(b ) ) . the process of data mining revealed the association between regulated genes unique to individual 
 treatment groups and certain biologic processes .
their correlation provided a better understanding 
 of underlying pathophysiology and a glimpse of key pathways for future focused studies .
one possible 
 application is the development of gene panels for genetic expression profiling as diagnostic tools . 

hierarchical clustering programs currently allow the generation of gene maps to be capable of 
 distinguishing between phenotypes .
in our simulation , highly regulated neurodevelopmental genes were 
 used and these successfully segregated treatments between microarrays ( figure 4 ) .
our findings strengthen our previous study 's assertion that fetal programming secondary to 
 maternal protein restriction renders an inherent susceptibility to neural compromise in offspring 
 and that the addition of dexamethasone to this vulnerable group results in further injury . in future 
 studies ,
the investigation of both sex - specific and transgenerational effects is necessary as 
 glucocorticoids influence endocrinological pathways differently in males and females .
also , timing 
 of exposure to glucocorticoids as well as dosage studies is no less relevant especially in the light 
 of reported evidence that a single course of therapy profoundly affects the fetal hpa axis [ 3335 ] . in conclusion
, the effects of antenatal steroid therapy can vary for each fetus according to 
 maternal - fetal factors and concurrent environmental stimuli .
further elucidating regulatory networks 
 that can mark the turning point between beneficial or damaging corticosteroid actions would result 
 in valuable adjustments of current treatment protocols .
the ability to recognize conditions highly 
 vulnerable to damage would also expand the possibility of tailored medicine more suitable to each 
 individual 's needs .
current biomolecular techniques are powerful tools in this field of study but 
 further validation between animal and true clinical models is required .
 

 introduction . 
 prenatal programming secondary to maternal protein restriction 
 renders an inherent susceptibility to neural compromise in neonates and any addition of 
 glucocorticosteroids results in further damage . 
 this is an investigation of consequent global gene 
 activity due to effects of antenatal steroid therapy on a protein restriction mouse model 
 . 
 methods . 
 c57bl/6n pregnant mice were administered control or protein restricted 
 diets and subjected to either 100  g / kg of 
 dexamethasone sodium phosphate with normosaline or normosaline alone during late gestation 
 ( e10e17 ) . 
 nontreatment groups were also included . 
 brain samples were collected on embryonic 
 day 17 and analyzed by mrna microarray analysis . 
 results . 
 microarray analyses 
 presented 332 significantly regulated genes . 
 overall , neurodevelopmental genes were overrepresented 
 and a subset of 8 genes allowed treatment segregation through the hierarchical clustering method . 
 the addition of stress or steroids greatly affected gene regulation through glucocorticoid receptor 
 and stress signaling pathways . 
 furthermore , differences between dexamethasone - administered 
 treatments implied a harmful effect during conditions of high stress . 
 microarray analysis was 
 validated using qpcr . 
 conclusion . 
 the effects of antenatal steroid therapy vary in 
 fetuses according to maternal - fetal factors and environmental stimuli . defining the key regulatory 
 networks that signal either beneficial or damaging corticosteroid action would result in valuable 
 adjustments to current treatment protocols . 
 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion 