Transcriptional Analysis Of Placentas From Large White Pigs And Erhualian Pigs On Later Gestation And Imprinting Detection

Chinese indigenous pigs are the most prolific breed of swine in the world.In fact, Large White pigs had no significant difference in number of live embryos with the Meishan pigs between 6-10 days after mating,but had significant lower litter size.One important reason of this phenomenon is associated with the placental efficiency difference in different breeds.During the later gestation stages,the Meishan pigs increased the vascular density,while the Large White pigs have the second increase in the area of placenta instead of vascular density,e.g,the placental efficiency between the two breeds are different.Thus,it is very important to identify molecular machenisms affecting placental efficiency to offer new ideas to improve reproduction traits in the pig.Imprinted genes are the genes whose expression is dependent on parental origin that is only one allele from the father or mother is expressed and the other allele is not expressed or lowly expressed.They play important roles in the fetal and placental development,maternal behavior and the postnatal growth.Most of the imprinted genes are expressed highly and imprinted in the placenta.At present,most studies on genomic imprinting are in human and mouse but little in livestock,especially in the pig.Therefore, it is of interest to identify more imprinted genes in pig placentas for analyzing the conservation of genomic imprinting among different species.In this study,twelve Erhualian(sub-population of the Taihu pigs) and Large White pregnant sows were necropsied at two ages respectively(75-and 90-d fetal,3 sows at each stage).The uteruses were removed immediately and the placentas were collected. Two female placentas from each sow were used to extract RNA and then pooled in equal quantity.The RNAs were sent to a commercial service for hybridization to the porcine Affymetrix GeneChip.Normalized data was used to analysis by GLM(SAS) model. Differentially expressed genes were identified and used to do GO(Gene Ontology) analysis,cluster and pathway analysis.Employing the RT-PCR-RFLP and product sequencing,we detected the allelic expression of eleven potential imprinted genes,and determined the imprinting status of these genes in the porcine placenta.The main results are as follows: 1.The transcriptome analysis indicated that 11,849 probesets were expressed in the placenta;921(p＜0.05) transcripts that were differentially expressed between E75 and L75;1145(p＜0.05) transcripts that were differentially expressed between E90 and L90.2.GO analysis indicated that the proteins encoded by these differentially expressed genes are associated with metabolism,fetal and placental development,blood vessel development and immunity processes.Cluster analysis showed that L75 and L90 were initially clustered together because their expression profiles were similar,E75 and E90 were clustered in another class.3.We selected eight genes(ALDH1A1,DIO3,DIRAS3,PLAGL1,PON2,ASCL2,WIF1 and SLC38A4) to confirm the microarray data using real-time RT-PCR.Among these genes,DIRAS3,PON2,PLAGL1,DIO and ASCL2 genes are candidate imprinted genes.The results indicated that the expression patterns of eight genes are consistent with the microarray data.4.Employing the Real-time PCR,we identified the expression of genes of VEGF pathway(VEGF,VEGFR-1,VEGFR-2,VE-cadherin andβ-arrestin 2) in E75,L75, E90 and L90.The results indicated that four genes(VEGFR-1,VEGFR-2, VE-cadherin andβ-arrestin 2) are differentially expressed in placentas between breeds.5.We chose PEG1,PEG3,PEG5,PEG10,GATM,INPP5F,PON2,DCN,PLAGL1, SLC38A4,DIRAS3 and ASCL2 genes as candidate imprinted genes in pigs according to the imprinting status in human and mouse and their biological functions.We obtained the complete open reading frame(ORF) of PEG1 and GATM genes with electronic cloning and sequencing.6.Using the RT-PCR-RFLP and sequencing,we detected the imprinting status of eleven genes in the porcine placentas.Seven genes(PEG1、PEG3、PEG5、PEG10、PLAGL1、SLC38A4 and DIRAS3) were imprinted and four genes(PON2,DCN,INPP5F and GATM) were not imprinted in the placenta.7.Using RH panel,we assigned PEG1,PEG3,PEG10,GATM and INPP5F genes to porcine 18q13-21,6q22-q23,9p13-21,1q12-21 and 14q29. 8.Using PCR-RFLP,we detected the allele frequencies of five SNPs in PEG1、PEG3、PEGIO,GATM and INPP5F genes among different populations.The different genotypes of HincⅡ-RFLP for INPP5F gene showed significant difference on the ADG from birth to 100Kg and ADG from 30 to 100Kg in the Large White pigs.9.Using Real-time PCR,we found that PEG5 gene is differentially expressed between E75 and L75,E90 and L90.The 1722 bp promoter sequence was successfully cloned.We predicated the structure and probable transcriptional factor sites of the promoter sequence and found some CTCF binding sites.The methylation of the CpG island in E75,L75,E90 and L90 were all high.