Gene Expression Profiles In Meishan And Yorkshire Swine Endometrium During The Early And Mid-gestation

The Chinese Meishan pig is well known for its high prolificacy compared to Western pig breeds. Reciprocal embryo transfers between Meishan and Large White pigs identified that the superiority of the Meishan sows for the trait litter size is solely determined by the maternal uterus genotype. The uterus holds the embryo and generates a histotroph rich environment to establish and maintain the pregnancy in pigs. In fact, the uterine function is controlled by number of complex molecules like hormones, cytokines and growth factors and their crosstalk. A network of these molecules plays a crucial role in preparing receptive endometrium and uterine capacity. In this study, we investigated the expression of genes in the purebred Meishan and Yorkshire sows endometrium on Days 15 of the estrous cycle, and Days 15,26 or 50 of gestation (n=2 pigs/day/status). We analyzed differentially expressed genes using a general linear model in SAS (SAS Institute, Cary, NC) for every gene separately.1 The global expressed profiling in Meishan and Yorkshire endometriumThe transcriptome analysis indicated that a total of 3001 transcripts were differentially expressed (FC>2 and FDR<0.05); 865 transcripts that were differentially expressed between M15 and M26; 2125 transcripts that were differentially expressed between Y15 and Y26; 150 transcripts that were differentially expressed between M26 and M50; 200 transcripts that were differentially expressed between Y26 and Y50; The results indicated that the uterine environment is much more different between the Meishan and Yorkshire sows during the early gestation than the mid-gestaion. The changes of the uterine environment of Meishan sows are more gradual compared with Large white pigs at the early gestation.2 Cluster analysisThe K-means cluster analysis results show that the specific expressed genes at day 15 of pregnancy is the calcium binding proteins which involved in the transport pathway, indicating these genes may be the new candidate markers for endometrium receptivity; The specific expressed genes at day 26 of pregnancy is the protease inhibitors which may inhibit the excessive invasion of trophoblast. And the is the positive and negative regulators of angiogenesis are highly expressed genes at days 26 and 50 of pregnancy. We selected six genes (HMOX1, MMP7, THBS1, HSD17B2, S100A9, PRLR, STC1, IGFBP3, ITGB3) to confirm the microarray data using real-time RT-PCR. The results indicated that the expression patterns of six genes are consistent with the microarray data.3 Secreted protein-encoded genes expression profiling in Meishan and Yorkshire endometriumSince the secretary activity of uterine is vital for the implantation and development of embryo and fetus, we identified the subcellular localization of the differentially expressed genes (DEGs) Using the UNIPROT web site, and potential porcine secreted protein genes were determined. The results indicated that the number of differentially expressed secreted protein coding genes between Y26 and Y15 is much more than differentially expressed secreted protein coding genes between M26 and M15, indicating the difference ability of uterine secretion may primarily emerge at the early pregnancy. GO analysis indicated that the up-regulated secreted protein coding genes involved in neuropeptide signaling pathway, G-protein coupled receptor protein signaling pathway and transforming growth factor beta receptor signaling pathway in M15 vs Y15. Analysis of Meishan and largewhite transcriptomes at gestation day 26 revealed that the biological processes are involved in regulation of growth and regulation of cytokine secretion. Analysis of Meishan and largewhite transcriptomes at gestation day 50 revealed that the biological processes are involved in blood vessel morphogenesis and regulation of growth. These data indicated that the endometrium receptivity of Meishan and Yorkshire sows may be different during the implantation stage, the fetal development in the Yorkshire endometrium may be faster than in the Meishan endometrium.4 Molecular characterization of the ISG15 and ISG43 geneThe real-time RT-PCR was performed to determine the expression profile of porcine ISG15 in the endometrium tissue at the gestation days 15,26 and 50 in Meishan and Yorkshire sows The differentially expression of porcine ISG15 and ISG43 in Meishan and Yorkshire endometrium during the early and mid-gestation indicated that porcine ISG15 may play important roles in the placentation and fetus development. The full-length of coding sequence of porcine interferon stimulated gene 15 and interferon stimulated gene 43 were cloned and their mRNA and deduced protein sequences’ characteristics were well defined. One ISG15 SNP (T171C) was assessed by HindⅢPCR-RFLP in almost 200 unrelated pigs, genotyping results showed great variation in allele frequency between Chinese indigenous and introduced commercial breeds. The polymorphic sites were also genotyped in the experimental pig population, the results showed that HindⅢ polymorphism in coding sequence of ISG15 was associated with birth weight (WB), total number born (TNB) and number born alive (NBA). One ISG43 SNP (A953C) was assessed by BccⅠPCR-RFLP in the experimental pig population, the results showed that BccⅠpolymorphism in coding sequence of ISG43 was associated with number born alive (NBA).5 The transcriptional mechanism of the ISG15 and LIF genesThe levels of ISG15 mRNA were markedly induced after 48 h in Ishikawa cells treated with E2 or E2 plus P4 indicating the important roles of ISG15 genes in the placentation. In order to identify their cis-regulatory elements, a series of deletion constructs from the 5’-flanking regions were analyzed. For ISG15, a construct containing a putative IRF1 and ISGF3 sites demonstrated the highest promoter activity levels. And a putative P53 site is located in the core region of LIF promoter.