Histological And Transcriptome Analysis Of Endometrium From Yorkshire And Meishan Sows

The higher embryo survival rate lead to the higher litter size in Meishan sows compared with Yorkshire sows.But the reason of its high embryo survival rate is still unknown.So the difference between Meishan pigs and Yorkshire pigs provide a precious animal model for study the mechanism of the embryonic survival.The transcriptome sequencing（RNA-Seq）can detect and analyze the whole transcriptional activity of cells or tissues,discover the novel transcripts and single nucleotide polymorphism（SNP）,recognize the alternative splicing sites and identify the different spliceosome at the same time which provide a comprehensive information of transcriptome.T This study used the Meishan and Yorkshire sows uterus at pregnant 18 and 32 days as the research object.Firstly,we maked the paraffin section to observe the morphological structure of the uterus organs by HE（hematoxylin-eosin）staining,and compared different organs under light microscope.Scondly,we analyzed the transcriptome of endometrium by RNA sequencing technology,and validated the sequencing results by the real-time fluorescent quantitative PCR,and identify the differentially expressed genes of endometrium in different breeds.Lastly,we collected the peripheral blood from Meishan and Yorkshire sows at different pregnancy days for content determination of estrogen and progestogen.The results are shown as follows:1.The Yorkshire uterine epithelia were simple columnar cells at pregnant 18 days,but it’s the false stratified columnar cells in Meishan uterine epithelia.At pregnancy 32 days,the Yorkshire uterine epithelial were double columnar cells,but in Meishan pigs it was double cuboidal cells.Moreover,the Yorkshire uterine glands are serous,but in Meishan pigs it emerged some mucous uterine glands besides serous uterine glands.We also measured the diameter and volume of uterus gland by Image pro-plus software.It demonstrated that the diameter at the pregnant 18 days was less than that at pregnant 32 days（P<0.05）.2.ELISA was used to measure the estrogen and progesterone concentrations of Meishan and Yorkshire sows at pregnant 9 days,12 days,15 days,18 days,25 days and 32 days.It was showed that Meishan serum estrogen concentrations in all period is significantly higher than the concentration of Yorkshire estrogen（P<0.05）.But before 18 days in pregnancy,progesterone concentration of Meishan pigs was significantly higher than that of Yorkshire pigs（P<0.05）,and there was no significant difference after 18 days in pregnancy.3.We respectively obtain 185715299 and 198390212 clean reads from endometrium of Meishan and Yorkshire sows by transcriptome sequencing;and there were 178409362 and 189763831 reads mapping to the porcine reference genome.The results of bioinformatics analysis indicated that there were 360 differentially expressed genes between Meishan and Yorkshire sows at pregnant 18 days,and 218 differentially expressed genes at pregnant 32 days.It had 1889 differentially expressed genes in day18-pregnant Meishan sows compared with day 32-pregnant Meishan sows.And in Yorkshire sows,the number was 1848.Three differentially expressed genes（XLOC₁250320、XLOC₂017489、XLOC₃15655）were choosed to verified by real-time fluorescent quantitative PCR analysis.All the results were consistent with the sequencing data,which indicated the accuracy of the sequencing.The high endometrial expression of the three genes implied that they may play important roles in the endometrium.4.We cloned the 2442bp 5’ flanking sequences of porcine XLOC 2017489 gene and found five potential SNP loci.The result of online software PROMO analysis showed that this sequences contained some transcription factor binding sites,such as C/EBPα、C/EBPβ、HOXA3、ER-α、STAT5A、PB-B、P53 and Spl.Some of them such as C/EBPa and C/EBPβ have multiple specific binding sites.5.Nine reporter plasmids of serial deletion fragments of porcine XLOC₂017489 promoter were constructed（Q1-Q9）and transfected into PK cell and HeLa cell to detect the relative activities by dual luciferase assay.The results showed that the core promoter of porcine XLOC₂017489 was located at-212～+15.