| The litter size is one of the important factors influencing the efficiency of pig production,and the fetal loss during pregnancy will greatly affect the litter size.The uterine lumina is spatially divided into the mesometrial side and the anti-mesometrial side.The mesometrial side of uterus is directly connected to the mother and is responsible for delivering oxygen and nutrients to the conceptus.Most fetal loss occur between gestational days 12-18 in pigs,during which time the pig conceptus undergo elongation and move to the mesometrial side of endometrium for mesometrial-biased implantation.Therefore,the mesometrial side and anti-mesometrial side of endometrium will respond differently to the mesometrial-biased implantation.In this study,meishan gilts were taken as research object during early pregnancy.First of all,the uterus were collected to make HE stained sections for morphological observation,and then the mesometrial side and anti-mesometrial side of endometrium on gestational days 12 and 15 were collected for RNA-seq and miRNA-seq.We identified spatiotemporal differentially expressed m RNAs,miRNAs and lncRNAs from the sequencing data.Through integrated analysis of the three sets of data,the interaction regulatory networks of lncRNA-miRNA-m RNA affecting the mesometrial-biased implantation were identified,and preliminary verification was carried out.The specific results were as follows:1. Identification of mesometrial-biased implantation phenotype during pig early pregnancy.With the help of the whole slide imaging system to scan the HE staining slides,we found that compared with the estrus uterus,the endometrium during implantation period were interlocked to close the uterine cavity,thus limiting the range of conceptus movement to the mesometrial side of endometrium.On gestational day 12,the floating conceptus migrated toward the mesometrial side of the uterus,but attachment had not been established with the endometrium.On gestational day 15,the degree of uterine luminal closure was deepened,and the further extended filamentous conceptuse was in contact with the uterine endometrium at the mesometrial side.2. Screening and functional analysis of genes related to the mesometrial-biased implantation in the pig endometrium during implantation period.Transcriptome analysis revealed that a total of 1753 genes were differentially expressed between gestational days 12 and 15 in the mesometrial endometrium,while a total of 1535 genes were differentially expressed between gestational days 12 and 15in the anti-mesometrial endometrium.At the same time,we revealed that 5 genes were differentially expressed between the mesometrial side and anti-mesometrial side of endometrium on gestational days 12,and 240 genes were differentially expressed between the mesometrial side and anti-mesometrial side of endometrium on gestational days 15.These results indicated that in addition to a large number of genes that were differentially expressed between gestational days 12 and 15,there were also a large number of genes that were differentially expressed between the mesometrial side and anti-mesometrial side of endometrium,which participated in the process of regulating mesometrial-biased implantation.And chemokines and their receptors(CXCL9,CXCL10,CXCL11,CXCL12,CXCL14,CXCR3,CXCR4,CXCR7),integrins and their related genes(ITGB2,ITGAL,ITGAM,ICAM1),selectin and its ligands(SELL,SELE,SELP,CD34)and other genes related to immune cell recruitment were up-regulated in the mesometrial side of the endometrium on gestational days 15.These spatial differentially expressed genes might regulate the mesometrial-biased implantation by means of biased recruitment of immune cells.3. Screening and functional analysis of miRNAs regulating genes which related to the mesometrial-biased implantation in the pig endometrium during implantation period.MiRNAome data revealed that a total of 45 miRNAs were differentially expressed between gestational days 12 and 15 in the mesometrial endometrium,while 41 miRNAs were differentially expressed between gestational days 12 and 15 in the anti-mesometrial endometrium.At the same time,we found that there was no differentially expressed miRNA between the mesometrial side and anti-mesometrial side of endometrium on gestational days 12.On gestational days 15,there were 17differentially expressed miRNAs between the mesometrial side and anti-mesometrial side of endometrium.By integrating the data of RNA-seq and miRNA-seq,we found that ALCAM-mi R-142-3p and STMN1-mi R-101 were differentially expressed between gestational days 12 and 15 and had a reverse expression pattern,which had potential regulation relationship.CXCL11-mi R-9,CXCR4-mi R-9,ITGB8-mi R-142-3p,which were differentially expressed between the mesometrial side and anti-mesometrial side of endometrium and had a reverse expression pattern,also had potential regulation relationship.Using the dual luciferase reporter system,we verified the potential binding relationships between these miRNAs and genes.These results indicated that miRNAs also had a spatiotemporal specific expression pattern similar to genes in the mesometrial side and anti-mesometrial side of endometrium on gestational days 12 and15,and might participate in the regulation of mesometrial-biased implantation by regulating their target genes.4. Screening and functional analysis of lncRNAs regulating genes which related to the mesometrial-biased implantation in the pig endometrium during implantation period.By identifying and analyzing the lncRNAs in strand specific RNA-seq data of the mesometrial side and anti-mesometrial side of endometrium on gestational days 12 and15,we revealed that 193 lncRNAs were differentially expressed between gestational days 12 and 15 in the mesometrial endometrium,while 210 lncRNAs were differentially expressed between gestational days 12 and 15 in the anti-mesometrial endometrium.At the same time,we revealed that 14 lncRNAs were differentially expressed between the mesometrial side and anti-mesometrial side of endometrium on gestational days 12,and 49 lncRNAs were differentially expressed between the mesometrial side and anti-mesometrial side of endometrium on gestational days 15.By analyzing the co-expression patterns of m RNAs and lncRNAs,combining the positional relationship between lncRNAs and m RNAs with the reverse expression pattern of miRNA and target genes at the same time,we revealed the regulatory interaction network of miRNA-m RNA-lncRNA in the mesometrial side and anti-mesometrial side of endometrium on gestational days 12 and 15.In addition,by predicting the candidate lncRNA that might function as ce RNA,we found that GTF2H1-AS,CNOT2-AS,and TCNOS?00080623 had potential binding sites for mi R-9.And these lncRNAs and mi R-9 had a reverse expression pattern between the mesometrial side and anti-mesometrial side of endometrium on gestational days 15.Then we used the dual luciferase reporter system to verify the regulatory relationship between lncRNAs and miRNA.These results indicated that lncRNA also had a spatiotemporal expression pattern in the mesometrial side and anti-mesometrial side of endometrium on gestational days 12 and 15 similar to genes,and lncRNA might interact with genes or endogenously compete for miRNA binding to affect the process of mesometrial-biased implantation.5. Screening candidate pathways related to mesometrial-biased implantation in the pig endometrium during implantation period.By integrating the data of lncRNA-miRNA-m RNA,we speculated that GTF2H1-AS,CNOT2-AS and TCNOS?00080623,which were up-regulated in the mesometrial side of endometrium on gestational days 15,could be endogenously competitive bounding mi R-9 as ce RNA.This resulted in the up-regulation of CXCL11 and CXCR4in the mesometrial side of endometrium on gestational days 15,which were the target genes of mi R-9.This regulatory relationship would eventually result in a higher concentration of chemokines in the mesometrial side of endometrium.More immune cells would be recruited to the mesometrial side of endometrium with a higher concentration of chemokines,which ultimately provided an important guarantee for the mesometrial side of endometrium to achieve an immune balance state to accept mesometrial-biased implantation.In addition,we also verified the phenomenon that more CD3~+immune cells were recruited to the mesometrial side of endometrium on gestational days 15 through the immunohistochemistry results.6. Association of SNPs in genes related to mesometrial-biased implantation with sow reproductive traits.We selected 6 genes(CXCR4,CXCL11,ALCAM,STMN1,ITGB8,MMP8)from the candidate pathways related to mesometrial-biased implantation,and identified 8SNPs in these 6 genes.By genotyping these SNPs in the Danish Yorkshire pig population and associating with the reproductive traits(total number born,total number born alive,effective number of born alive,litter size at day 5,mummy,stillbirth,gestation period,parity interval),we found that 8 SNPs in these 6 genes were associated with reproductive traits.These results indicated that these SNPs could be used as potential molecular markers for sow reproductive traits selection in breeding practices. |
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