animal and treatment : one week after the artificial insemination , second parity landrace  
 yorkshire crossbred sows were randomly divided into two groups . sows in control group ( n = 6 ) were fed with 
 standard diet ( table 1table 1.composition and nutrient content of the experimental dietcontrolbetaineingredient , g / kgcorn370370wheat300300bran8080soybean meal170170lignocelluloses3030cahpo42020soybean oil88premix*2020betaine03digestible energy , mj / kg13.113.1calculated compositioncrude protein , % 1515crude fiber , % 4.54.5calcium , % 0.840.84phosphorous , % 0.650.65 * the premix contains ( per kg ) : vitamin a : 240,000 iu ; vitamin d3 : 60,000 iu ; vitamin e : 720 iu ; vitamin 
 k3 : 30 mg ; vitamin b1 : 30 mg ; vitamin b2 : 120 mg ; vitamin b6 : 60 mg ; vitamin b12 : 360 mg ; niacin : 600 mg ; 
 pantothenic acid : 300 mg ; folic acid : 6 mg ; manganese sulphate : 1.0 g ; zinc oxide : 2.5 g ; iron sulphate : 
 4.0 g ; copper sulphate : 4.0 g ; sodium selenite : 6 mg ; calcium : 150 g ; phosphorus : 15 g ; sodium chloride : 
 40 g. ) , and those in betaine group ( n = 6 ) were fed with diet supplemented with 3 g / kg betaine 
 hydrochloride of 98% purity ( skystone feed co. , ltd . ,
all 
 animals were treated in the same way of insemination and reared under the same housing condition .
the hippocami 
 of male offspring ( one per litter ) were sampled at delivery , immediately frozen in liquid nitrogen and stored at 
 80c . *
the premix contains ( per kg ) : vitamin a : 240,000 iu ; vitamin d3 : 60,000 iu ; vitamin e : 720 iu ; vitamin 
 k3 : 30 mg ; vitamin b1 : 30 mg ; vitamin b2 : 120 mg ; vitamin b6 : 60 mg ; vitamin b12 : 360 mg ; niacin : 600 mg ; 
 pantothenic acid : 300 mg ; folic acid : 6 mg ; manganese sulphate : 1.0 g ; zinc oxide : 2.5 g ; iron sulphate : 
 4.0 g ; copper sulphate : 4.0 g ; sodium selenite : 6 mg ; calcium : 150 g ; phosphorus : 15 g ; sodium chloride : 
 40 g. operating procedures for animal breeding and husbandry were in accordance with technical regulations for 
 commercial pig production for intensive pig farms ( gb / t 17824.2 - 2008 ) .
the experimental protocol was approved by 
 the animal ethics committee of nanjing agricultural university , with the project number 2012cb124703 .
the 
 slaughter and sampling procedures complied with the  guidelines on ethical treatment of experimental animals  
 ( 2006 ) no . 398
after pretreated with rnase - free dnase , 2 g of total rna was reverse - transcribed to cdna in 
 obedience to the protocol provided in the random hexamer primers kit ( promega , madison , wi , u.s.a . ) .
two 
 l of diluted cdna ( 1:25 , vol / vol ) was used for real - time pcr which was performed with a 
 mx3000p real - time pcr system ( stratagene , santa clara , ca , u.s.a . ) .
all the primers , synthesized by generay 
 biotech , for determining gr total mrna and its variants and internal control ( -actin ) are listed in table 2table 2.primers for real - time pcr amplification of gr mrna and its variants , segments of gr promotertarget geneproduct lengthprimer sequencereference(bp)(f : forward , r : reverse)gr108f : 5-ccaaactctgccttgtgtgttc-3ay779185r : 5- tgtgctgtccttccactgct-3gr 1 - 4161f : 5-cacacagcacaacctttc-3r : 5-aaccttcacaggagttcc-3gr 1 - 5207f : 5-gcgtgcaacttccttcaa-3r : 5-cttggagtctggctgaga-3gr 1 - 6189f : 5-gagtgggccgcccagacgat-3r : 5-ccccccctcaggcttttat-3gr 1 - 7185f : 5-gcgaagagaaactagagaaa-3r : 5-aaccttcacaggagttcc-3gr 1 - 8144f : 5-tgcccagcgtcgccaaca-3r : 5-ccgcccctcaggcttttat-3gr 1 - 9,10177f : 5-cctgctttcacacgctaa-3r : 5-atcacatgggctctctcc-3gr 1 - 11163f : 5-ctggtggaagtgggcgtgtc-3r : 5-ttcctcccctcaggcttttat-3-actin201f : 5-cccacggaatcgagaaagag-3af057040r : 5-ttgacggaagggcacca-3gr segment1f : 5-cggcgaaggtctaggtacg-3r : 5- gaaggctgccccgtgt-3gr segment2f : 5- tctttgaaccccgcactt-3r : 5- ctcccagcgacaaaccag-3negative controlf : 5- ctgggcatcagaacctgt-3(actb promoter)r : 5- gagcaatcccctgaagaa-3. data analysis conformed to the method of 2 .
the abundance of gr variants mrna was presented as the percentage of total gr mrna in control 
 group .
protein samples , after being measured concentrations with a pierce bca protein assay kit ( no . 23225 , thermo , 
 rockford , il , u.s.a . ) , were denatured in waterbath for 5 min at 100c and seperated in a 7.5% or 10% sds - page . 
 after protein transfer , nitrocellulose membranes ( biotrace , pall co. , ann arbor , mi , u.s.a . )
were blocked with 
 4% bsa and incubated respectively with anti - gr ( sc-1004 , santa cruz biotechnology , dallas , tx , u.s.a . , 1:500 ) 
 and anti - actb ( ap0060 , bioworld , atlanta , ga , u.s.a . , 1:10,000 ) antibodies overnight at 4c .
bands were 
 visualized by enhanced chemiluminescence with the lumiglo substrate ( super signal west pico trial kit , pierce , 
 rockford , il , u.s.a . ) and captured by versadoc 4000mp system ( bio - rad , hercules , ca , u.s.a . ) to calculate the 
 value of band density using quantity one software ( bio - rad ) automatically .
methylated dna immunoprecipitation analysis : hippocampal genomic dna was sonicated into 
 fragements approximately 500 bp in size .
two g of sonicated dna were denatured and incubated 
 with the antibody against 5-methyl cytosine ( ab10805 , abcam , cambridge , ma , u.s.a . ) to immunoprecipitate the 
 methylated dna fragments . then
, protein g agarose beads were used to capture the dna - antibody complex which was 
 washed and treated with proteinase k to release the precipitated dna .
the medip dna was then extracted , purified 
 and used to amplify the fragment of gr promoter by real - time pcr , following the procedure described previously 
 .
the promoter region of actb gene containing no cpg sites was 
 amplified to serve as a negative control in medip analysis .
data were normalized against the negative control and 
 presented as the fold change relative to the average value of the control group .
prediction and determination of mirnas : the potential mirnas targeting the 3 utr of gr mrna 
 were predicted with the probability of interaction by target accessibility ( pita ) algorithm   based on the online database of pig micrornas ( http://www.mirbase.org/ ) , 
 using microrna prediction tool ( http://genie.weizmann.ac.il/pubs/mir07/mir07_prediction.html ) .
two g of total rna from each piglet were polyadenylated by poly(a ) polymerase ( pap ) at 37c 
 for 1 hr using a poly(a ) tailing kit ( am1350 ; ambion , rockford , il , u.s.a . ) , according to the manufacturer s 
 instructions .
after phenol / chloroform extraction and ethanol precipitation , treated rna was then dissolved and 
 reverse - transcribed using poly(t ) adapter and m - mlv ( promega ) .
real - time pcr was performed in an mx3000p 
 ( stratagene ) with sybr_premix ex taqtm ii ( takara , otsu , japan ) using a mirna - specific forward primer and a 
 universal reverse primer complementary to part of the poly ( t ) adapter sequence .
the sequences for all the primers and the poly ( t ) adapter are 
 listed in table 3table 3.primers for real - time pcr amplification of mirnasprimerprimer sequencemir-18b5-taaggtgcatctagtgcagttag-3mir-22 - 3p5-aagctgccagttgaagaactgt-3mir-22 - 5p5-agttcttcagtggcaagcttta-3mir-30a-3p5-ctttcagtcggatgtttgcagc-3mir-30a-5p5-tgtaaacatcctcgactggaag-3mir-130b5-cagtgcaatgatgaaagggcat-3mir-1385-agctggtgttgtgaatcaggc-3mir-181a5-aacattcaacgctgtcggtgagtt-3mir-1835-tatggcactggtagaattcactg-3mir-181d5-cccaccgagggatgaatgtcac-3mir-181c5-aacattcaacctgtcggtgagt-3universal reverse primer 5-tagagtgagtgtagcgagca-3u65-ggcaaggatgacacgcaaat-3poly ( t ) adapter 5-tagagtgagtgtagcgagcacagaattaatacg actcactataggttttttttttttttttvn-3. the abundance of mirnas was presented as the fold change relative to the average value of the 
 control group .
functional validation of mir-130b and mir-181d : dual luciferase activity assay was used to 
 verify the function of mir-130b and mir-181d targeting gr 3utr in vitro , as previously 
 described .
briefly , fragments of mir-130b and mir-181d precursor and 
 the scramble control ( sc ) sequences ( table 4table 4.nucleotides sequences used in functional validation of mirnasnamesequences ( f , forward ; r , reverse)mir-130b precursorf:5-gatccccttggcataacgtagcagcacataatggtttgtgggttttgaaaaggtgcaggccatattgtgctgcctcaaaaatacaaggttttttggaaa-3r:5-agcttttccaaaaaagcctgactgatgccctttcatcattgcactgcttcccagtggcccacagtagtgcaacagggaaagagtgtcaggcaggcg-3mir-181d precursorf:5-tgctgcccaccgagggatgaatgtcacgttttggccactgactgacgtgacatttccctcggtggg-3r:5-cctgcccaccgagggaaatgtcacgtcagtcagtggccaaaacgtgacattcatccctcggtgggc-3mir - scf:5-gatccgacttacagccagttcctagtatagtgaagcagcagatggtatactaggaactggctgtaagctttttttggaaa-3r:5-agcttttccaaaaaaagcttacagccagttcctagtataccatctgctgcttcactatactaggaactggctgtaagtcg-3gr 3utr primerf:5-tctagactttcgttggtgtat-3r:5- tctagagcaaacccattggg-3 ) were subcloned to construct plasmids named psilence - mir-130b , psilence - mir-181d and 
 psilence - mir - sc , respectively ( fig . 3afig .
1.rt-pcr demonstrated that hippocampal expression of gr exon 1 - 4 and 1 - 9,10 mrna was higher in 
 betaine - treated group , associated with the higher total gr mrna .
meanwhile , western blotting analysis 
 showed that total gr protein content was not changed so much .
values are means , with their standard 
 errors , and * indicates significant difference between groups at p<0.05 , * * means at 
 p<0.01.fig .
2.cpg sites are underlined in detectable segment1 ( 4861 ~ 4722 ) and segment2 ( 1650 ~ 1515 ) from 
 translation start codon ( atg ) , which were marked as block in gr exon 1 structure diagram ( blank arrow ) . 

medip results are presented in 
 column figure , showing dna methylation status of cpgs in segment2 was increased .
values are means , with 
 their standard errors , and * indicates significant difference between groups at 
 p<0.05.fig .
( c ) statistic results of the data from dual luciferase activity 
 assay system .
* * indicate significant difference 
 between groups at p<0.05 and p<0.01 , respectively . ) .
gr 3 utr sequence was amplified by pcr using the specific primers ( table 4 ) , and the product was then subcloned to pgl3-control 
 using xbai ( invitrogen , rockford , il , u.s.a . ) to construct the pgl3-gr - utr plasmid ( fig .
hela cells were transfected with the plasmids with an electroporation device , 
 and prl - tk was used to normalize the transformation efficiency .
after 24 hr incubation at 5% co2 and 
 37c , firefly and renilla luciferase activities were measured , and the targeting efficacy of mirnas in 
 post - transcriptional repression of reporter protein was presented as the fold change relative to the average 
 value of the psilence - mir - sc .
rt - pcr demonstrated that hippocampal expression of gr exon 1 - 4 and 1 - 9,10 mrna was higher in 
 betaine - treated group , associated with the higher total gr mrna .
meanwhile , western blotting analysis 
 showed that total gr protein content was not changed so much .
values are means , with their standard 
 errors , and * indicates significant difference between groups at p<0.05 , * * means at 
 p<0.01 .
cpg sites are underlined in detectable segment1 ( 4861 ~ 4722 ) and segment2 ( 1650 ~ 1515 ) from 
 translation start codon ( atg ) , which were marked as block in gr exon 1 structure diagram ( blank arrow ) . 
 predicted transcription factor binding sites among segment2 were framed .
medip results are presented in 
 column figure , showing dna methylation status of cpgs in segment2 was increased .
values are means , with 
 their standard errors , and * indicates significant difference between groups at 
 p<0.05 .
( b ) plasmid structure containing gr 3utr sequences . cloning 
 region is indicated in dot line with gr 3utr sequences ( 1,384 bp ) .
( c ) statistic results of the data from dual luciferase activity 
 assay system .
statistical analysis : all data were expressed as means  sem and analyzed with one - way anova 
 for independent samples with statistical packages for the social sciences ( spss ) 11.0 for windows .
expression of total gr mrna and the alternative exon 1 mrna variants , as well as the total protein 
 content of gr : as shown in fig .
1 , the hippocampal 
 expression of total gr mrna was significantly higher ( p<0.01 ) in offspring piglets born to 
 betaine - treated sows .
seven alternative gr exon 1 mrna variants were detected in the hippocampus of newborn 
 piglets .
but , only gr 1 - 4 ( p<0.05 ) and gr 1 - 9,10 ( p<0.01 ) mrna variants 
 were significantly upregulated in betaine group .
nevertheless , no significant alteration was observed in total 
 gr protein content as revealed by western - blot analysis ( fig .
1 ) . methylation level of gr promoter sequences : two segments of gr promoter , 4861 ~ 4722 and 
 1650 ~ 1515 from translation start codon ( atg ) , were analyzed using medip assay .
the sequences of these two 
 segments and the position relative to gr exon 1 variants are shown in fig . 
 2 .
data from medip analysis indicated that the methylation level of segment 1 ( 4861 ~ 4722 ) was not 
 significantly changed , but segment 2 ( 1650 ~ 1515 ) was hypermethylated ( p<0.05 ) in betaine 
 group ( fig .
the transcription factor prediction analysis with 
 patch ( http://www.gene-regulation.com/cgi-bin/pub/programs/patch/bin/patch.cgi ) showed many 
 potential transcription factor binding sites within this segment ( fig . 
 2 ) .
expression of mirnas targeting gr : the abundance of mir-181a ( p<0.05 ) , 
 mir-181d ( p<0.01 ) and mir-130b ( p<0.05 ) was significantly higher in the 
 hippocampus of betaine - treated piglets compared to control counterparts ( table 5table 5.expression of mirnas targeting porcine gr 3utrpredicted mirnascontrol ( n = 5)betaine ( n = 5)p - valuemir-130b1.00  0.091.35  0.090.03 mir-1381.00  0.061.10  0.060.26 mir-18b1.00  0.091.09  0.100.50 mir-181a1.00  0.061.22  0.060.04 mir-181c1.00  0.081.07  0.090.61 mir-181d1.00  0.413.08  0.140.00 mir-1831.00  0.131.29  0.140.16 mir-22 - 3p1.00  0.061.07  0.090.51 mir-22 - 5p1.00  0.101.10  0.110.54 mir-30a-3p1.00  0.091.04  0.050.71 mir-30a-5p1.00 
no significant difference was detected for the expression of mir-18b , 22 - 3p/5p , 30a-3p/5p , 138 , 
 183 or 181c between control and betaine - treated groups .
functional validation of mir-130b and mir-181d : the schematic maps of the ssc - mir-130b and 
 ssc - mir-181d over - expression plasmids , as well as the luciferase reporter plasmid containing 1384 bp of pig gr 
 3 utr , are shown in fig . 3a and 3b , respectively .
overexpression of 
 mir-130b ( p<0.05 ) or mir-181d ( p<0.01 ) was able to significantly reduce 
 the luciferase activity of hela cells transfected with the pig gr 3utr reporter plasmid , as compared with the 
 scramble control ( fig .
it is well - documented that gr expression can be modulated by maternal nutritional interventions , such as 
 protein restriction in rats [ 4 , 21 ] 
 and undernutrition in sheep , as well as dietary supplementation of 
 methyl donors in rats   and in humans .
moreover , infusion of l - methionine diminished hippocampal gr expression in the adult rat 
 offspring of high licking / grooming and arched - back nursing ( lg - abn ) mothers . similarly , maternal dietary supplementation with methyl donor mixture inhibited hippocampal gr 
 expression in three - month - old tame rats . in the present study , 
 however , maternal betaine supplementation increased total gr mrna expression in the hippocampus of newborn 
 piglets .
the diverse responses of hippocampal gr expression to maternal methyl donor supplementation may 
 attribute to multiple factors , such as the type and the dose of the substances , the timing and the duration of 
 supplementation , the species , genotype and the age of the animals investigated .
moreover , the distribution 
 patterns of gr exon 1 mrna variants differ significantly between species . in pigs , gr mrna variants , 1 - 4 , 1 - 6 
 and 1 - 9,10 ,
are abundantly expressed and tightly regulated in the hippocampus [ 29 , 31 ] , while in rats , gr 1 - 7 appears to be the most important 
 mrna variant of functional significance in hippocampus .
therefore , 
 it is presumed that the transcriptional regulation of gr expression in hippocampus is complex and 
 species - specific . in our experiment ,
significant change of hippocampal gr expression in mrna level was observed 
 with maternal betaine treatment , but not in protein level .
probably , potential participant of transcript 
 regulation , such as dna methylation and mirnas , was also induced by maternal betaine to maintain gr protein 
 homeostasis in the hippocampi of these piglets .
the effects of maternal methyl donors on the regulation of gr transcription often involve changes of the 
 methylation status in gr promoter regions .
higher choline intake led to hypermethylation of 5 untranslated exon 
 1f ( homologue of exon 1 - 7 in rats ) of gr promoter in human placenta , 
 while methionine treatment reversed the hypomethylation of the exon 1 - 7 gr promoter in the hippocampus of adult 
 rat offspring of high lg - abn mothers , as revealed with sodium bisulfite sequencing technique .
nevertheless , it was also reported that feeding methyl - supplemented diet 
  or methyl donor deficiency diet   to pregnant dams did not alter the methylation level of gr exon 1 - 7 promoter in the hippocampus of 
 rat offspring .
these results suggest that the maternal or prenatal supplementation of methyl donors has minor , 
 if any , effects on methylation status of gr exon 1 - 7 promoter in the hippocampus of rat offspring . in the 
 present study ,
only two segments in the region of porcine gr promoter were investigated , and the segment ( 1650 
 ~ 1515 ) was found to be hypermethylated in the hippocampus of maternal betaine - treated piglets .
the other 
 regions can not be amplified due to extremely high enrichment of cpgs .
certainly , more detailed and higher 
 resolution analysis of methylation pattern across the whole region of gr promoter is required , by using more 
 powerful techniques , to elucidate the role of cpg methylation in the transcription regulation of different gr 
 mrna variants .
it is well known that dna hypermethylation in gene promoter is functioned for suppression of mrna expression 
 when positive transcript factors can not bind to the corresponding sequences .
otherwise , binding loss of 
 negative transcript factor would contributed to promoting mrna expression , as we previously reported that 
 hypermethylation of differentially methylated regions in igf2 gene promoter was associated with high expression 
 of igf2 mrna . here
, we found that the segment ( 1650 ~ 1515 ) of gr 
 gene promoter was hypermethylated , which might co - work with negative transcript factors to improve gr mrna 
 expression .
the segment ( 1650 ~ 1515 ) is located upstream of the translation start codon ( atg ) in exon 2 , 
 which contains multiple transcription factor binding sites ( fig .
2 ) , 
 predicted to be involved in the transcriptional regulation of gr expression .
therefore , feeding 
 betaine - supplemented diet to pregnant sows modulated hippocampal gr transcription in neonatal offspring piglets 
 through , at least partly , alterations of methylation status in gr promoter .
gr expression is also regulated at the post - transcriptional level . in this study ,
the increased abundance of 
 total gr , as well as its 1 - 4 , and 1 - 9,10 mrna variants , did not lead to significant change in the level of gr 
 protein in the hippocampus of betaine - treated piglets .
we presumed that there might be mirna - mediated 
 post - transcriptional regulation involved .
methyl donor deficiency ( mdd ) in utero caused growth 
 retardation of rat fetus at embryonic day 20 associated with upregulated expression of the stat3 regulator 
 mir-124 .
we reported previously that feeding betaine - supplemented 
 diet to pregnant sows significantly altered hepatic expression of mir-497 and mir-181 in neonatal offspring 
 piglets . in this study , hippocampal expression of mir-130b , mir-181a 
 and mir-181d was significantly up - regulated in neonatal piglets of betaine group .
overexpression of mir-130b was 
 reported to decrease the expression of endogenous gr protein and the activity of the luciferase reporter 
 containing the 3utr of human gr mrna in gc - sensitive mm.1s cells . 
 in the present study ,
the suppressive function of ssc - mir-130b and ssc - mir-181d on gr expression was validated 
 in hela cells transfected with a luciferase reporter plasmid containing pig gr 3utr sequence .
mir-181d was 
 previously reported to target methyl - guanine - methyl - transferase gene in human   and was implicated to be a potential target for glioma therapy .
furthermore , mir-181d was found to be one of the stress - responsive mirnas which may target a 
 number of stress and metabolic signaling pathways in thymocytes . 

however , direct evidences to support the suppressive effect of mir-181d on gr expression are lacking . to our 
 knowledge , here
, we provide the first evidence that ssc - mir-181d is able to target porcine gr 3utr and suppress 
 the luciferase reporter activity in hela cells . in conclusion , we demonstrate , for the first time , that maternal betaine supplementation during gestation 
 enhances the expression of total gr mrna , as well as its exon 1 - 4 and 1 - 9,10 mrna variants , in the hippocampus 
 of neonatal piglets , without affecting the total cellular gr protein content .
alterations of cpg methylation in 
 the proximal region of gr promoter and modified expression of mirnas targeting 3utr of porcine gr mrna appear 
 to be involved in the regulation of hippocampal gr expression .
long - term follow - up studies are required to 
 evaluate the possible consequences of maternal betaine supplementation regarding the health and performance of 
 pigs in later life .
 methyl donor nutrients are critical for embryonic development of brain . 
 hippocampus is the most susceptible 
 brain region to various factors including prenatal supply of methyl donors . 
 glucocorticoid receptor ( gr ) 
 expressed in hippocampus is involved in the regulation of energy homeostasis and stress sensitivity . 
 
 hippocampal gr expression is highly susceptible to epigenetic regulation , yet the effect of maternal methyl 
 donor supplementation on epigenetic regulation of gr transcription in offspring hippocampus remains unclear . 
 in this study , we fed sows with betaine ( 3 g / kg ) throughout the gestation and analyzed the hippocampal 
 expression of gr mrna and its variants , as well as the cpg methylation status of the promoter and the 
 micrornas predicted to target 3 utr of porcine gr gene in neonatal piglets . 
 total gr mrna 
 ( p<0.01 ) and its variants gr 1 - 4 ( p<0.05 ) and 1 - 9,10 
 ( p<0.01 ) , were significantly higher in the hippocampus of betaine - treated piglets , while 
 the content of gr protein was not significantly changed . 
 the cpgs located in the 1650 ~ 1515 segment of gr 
 gene were hypermethylated ( p<0.05 ) . 
 the hippocampal expression of mir-130b 
 ( p<0.05 ) , mir-181a ( p<0.05 ) and mir-181d 
 ( p<0.01 ) was significantly up - regulated . 
 the targeting efficacy of mir-130b and mir-181d 
 was validated in vitro using dual - luciferase reporter assay system . 
 our results demonstrate 
 that maternal betaine supplementation during gestation enhances gr mrna expression in offspring hippocampus , 
 which involves alterations in mirnas expression . 