| The litter size of sows is one of the most important indexes to assess the fertility of sows in pig production.The loss of embryos in the early pregnancy of sows has a direct effect on litter size.The early embryo implantation stage of sows is the key stage of embryo loss.Embryo implantation is a dynamic developmental process involving a series of physical and physiological interactions between the embryo and the endometrium,which requires the development of the embryo and the establishment of the receptive state of the uterus.This is a complex physiological process,which is accurately regulated by diversified biological molecules including growth factors.In this study,the Yorkshire sows during preimplantation(days 9-11 of pregnancy),implantation period(days 12-14 of pregnancy),and post-implantation period(days 15 and 18 of pregnancy)were used as the research objects to perform a comprehensive analysis of the endometrial differentially expressed(DE)proteins profile through iTRAQ technology.In addition,we explored the effect of differentially expressed protein RBP4 on embryo implantation by RT-q PCR,CCK8 method,flow cytometry,mouse uterine horn injection,and other experimental techniques.This study provides a theoretical basis for understanding the molecular mechanism of porcine embryo implantation,and also provides resources for the protein research of endometrial tissue during the porcine embryonic implantation period.The main research results are as follows:1.The iTRAQ technique was used to analyze the differences in endometrium proteome during the implantation stage of porcine embryos.We used 8 samples with different pregnancy days(days 9-15 and 18 of pregnancy)as a set of iTRAQ test groups,and three replicates as three groups for iTRAQ.Among them,each group used the sample on day 9 of pregnancy as the internal control.The peptides and proteins(FDR adjusted P value<0.01)were selected.19,954 Unique peptides and 5444 proteins were obtained.We found that the expression of DE proteins such as S100A9,S100A12,APOC3,HRG,etc.using the MRM method are highly correlated with the measured value of iTRAQ.2.A total of 4779 differentially expressed proteins were found by comparing the protein expression levels in the porcine endometrium tissues on different gestation days.At the same time,the differentially expressed proteins patterns of Y9-Y15(Yorkshire pigs on days 9-15 of pregnancy)and Y18 were clustered,and it was found that the expression patterns of Y12 and Y13 were similar.In addition,the expression patterns of Y14 and Y15 were also similar.There was no direct clustering between Y10 and the other periods,indicating that the expression pattern of Y10 was significantly different from that of other periods.3.We performed GO,KOG,KEGG analysis of total protein(all identified proteins).The GO analysis indicated that total proteins were enriched in "binding","immune system process","localization","locomotion" and other GO terms.KOG analysis showed that the total proteins were related to "Signal transduction mechanisms","Transcription","Lipid transport and metabolism",etc.In KEGG analysis,total protein is mainly enriched in "Cell Growth and Death","Cell Motility",and "Immune System".4.Through GO analysis of the differentially expressed proteins in different periods,the results show that pre-implantation,implantation period,and post-implantation period DE proteins are significantly enriched in GO terms such as "binding","extracellular region".It is worth noting that Y12 vs Y9 and Y13 vs Y9 are both significantly enriched in the "defense response" GO term;this may be related to the immune tolerance of the endometrium during embryo implantation.KOG analysis of the differential proteins showed that the differential proteins in the pre-implantation,implantation period,and postimplantation period were mainly related to cytoskeleton,signal transduction,energy generation,and conversion,etc.KEGG analysis showed that: during pre-implantation,the DE proteins were mainly related to calcium channel and immune regulation.In addition,Y12 vs Y9 also enriched in the PI3K-Akt signaling pathway during the implantation period.The DE proteins during the post-implantation period were mainly related to immunity and metabolism.5.By predicting the subcellular localization of the different proteins,it was found that they were mainly localized in extr(extracellular component),cyto(cytoplasm),and nucl(nucleus)in the pre-implantation,implantation period,and post-implantation period.6.Through the functional study of RBP4,it was found that the knockdown of RBP4 inhibited the proliferation and migration of primary porcine endometrial epithelial and stromal cells,and promoted the apoptosis of both cells.In vivo injection experiments in mice showed that the interference of RBP4 in the uterus significantly reduce the embryo implantation rate.The above results indicate that RBP4 can affect embryonic implantation by affecting the receptivity of the endometrium. |
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