| Pig litter size is the affecting factor of pork production,which is an important part of national economics.One of key factors affecting pig litter size is the survival rate of embryo during the early implantation.The prerequisite for successful implantation in early pregnancy is the precise coordination between the embryo and the uterine components,such as uterine luminal epithelium,glandular epithelium,stroma and muscular layer.Porcine placenta type is epithelial chorionic type and pig embryo attaches directly to the uterine luminal epithelium until delivery.So uterine luminal epithelium cells act on uterus receptivity,placentation,exchange of signal,energy,materials between embryo and uterus during embryo implantation.From the aspects of anatomy,we can divide uterine epithelium cells into mesometrial side and anti-mesometrial side,and the prerequisite for successful embryo implantation is that pig embryo locates at mesometrial side of uterine cavity.Gene expression pattern of uterine luminal epithelium may be different between mesometrial side and anti-mesometrial side,which are involved in regulation of embryo implantation.So we collected many uterus tissue on ed12,ed15,gd12(before embryo implantation)and gd15(after embryo implantation).Firstly,an identification method of histology at the maternal-embryo interface during pig embryo implantation was constructed,which identified new histological phenotype related with pig embryo implantation;secondly,spatiotemporal transcriptome of pig uterine luminal epithelium cells-embryo and ATAC-seq of pig uterine luminal epithelium cells were obtained;Lastly,we identified important genes and regulation network related with pig embryo implantation.The main results are as follows:1 Embryo distribution characteristics in the uterus cavity during pig embryo orientation implantationAs spatial characteristics of pig embryo orientation implantation is the study basis that embryo attaches into the uterine epithelium,so we innovated the histological identification method for observing space characteristic in situ of pig embryo in the uterus cavity.We mainly found three types of histological phenotype at the maternal-embryo interface during pig embryo implantation:(i)conceptus located in mesometrial side,anti-mesometrial side,between mesometrial side and anti-mesometrial side,(ii)uterus cavity owns dynamic change phenotype "open-closure-open",(iii)more than one conceptus can exist in the same interface of the uterine cavity.These phenotypes show spatial characteristics of embryo specific location implantation are various and complex.Meanwhile,we established a pipeline of 3D reconstruction using small uterus segment.From the reconstruction of 3-dimension imaging,we can observe spatial structure in the uterus cavity which embryo implants.The identification of these characteristics provide new histological phenotype for studying embryo specific location implantation further.2 Spatiotemporal transcriptome analysis of pig uterine epithelium and embryo in the estrus and the period of embryo implantationIn order to study molecular characteristics of embryo specific location implantation in the maternal-embryo interface,we established single cells capture technology in situ based on laser capture microdisection.We obtained embryo and uterine epithelium cells at mesometrial side and anti-mesometrial at same interface of uterus cavity on ed12,ed15,gd12,gd15 and carried out RNA-seq.With the 11 comparison analysis results in different position(mesometrial side and anti-mesometrial side),different stages,estrus and gestation,we mainly found:(1)Gene expression of luminal epithelium cells exists significant difference between mesometrial side(embryo implantation position)and anti-mesometrial side during pig embryo implantation.Number of DEGs of uterine epithelium cells from the comparison between mesometrial side and anti-mesometrial side(345 DEGs on gd12 and596 DEGs on gd15)is more than that of estrus(3 DEGs on ed12 and none DEGs on ed15),and number of DEGs on gd15 is more than that on gd12.Pathway enrichment of DEGs above contains extracellular exosome,cell adhesion and intergrins-regulated signal pathway,which shows that uterus cavity is divided into different functional zone under the induction of embryo implantation signal as the gene expression difference of luminal epithelium cells between embryo implantation side and embryo non-implantation side,resulting in embryo implantation at the mesometrial side.(2)Gene expression of luminal epithelium cells has significant difference during the gestation.Number of DEGs of uterine epithelium cells at mesometrial side and anti-mesometrial side between gd12 and gd15(1699 DEGs at mesometrial side,1607 DEGs at mesometrial side)is more than that of uterine epithelium cells at mesometrial side and anti-mesometrial side between ed12 and ed15(53 DEGs at mesometrial side,93 DEGs at mesometrial side).Pathway enrichment of DEGs above mainly contains extracellular exosome,protein digestion and absorption,metabolic pathway,which shows functional difference of luminal epithelium cells is more significant between mesometrial side and anti-mesometrial side as the development of embryo implantation process.(3)Gene expression of calcium binding and corresponding pathway is different between the status of estrus and gestation.Number of DEGs of uterine epithelium cells between the status of estrus and gestation are 2007(mesometrial side between gd12 and ed12),1880(anti-mesometrial side between gd12 and ed12),1004(mesometrial side between gd15 and ed15)and 600(anti-mesometrial side between gd15 and ed15).Pathway of DEGs of luminal epithelium cells at mesometrial side and anti-mesometrial side between estrus and gestation mainly enriched in calcium binding,cellular calcium response,positive regulation of release of sequestered calcium ion into cytosol and cellular calcium ion homeostasis,which shows calcium ion homeostasis plays a role in the process of embryo specific location implantation.3 Identification of candidate genes and transcription factors related with pig embryo implantation on uterine epithelium cells(1)Identification of key genes affecting pig embryo specific location implantation.In order to identify key candidate genes related with embryo specific location implantation,we do this from two aspects based on transcriptome we obtain from maternal-embryo interface.First,we screened four DEGs(SPP1?SULT2A1?THY1?MEP1B)between mesometrial side and anti-mesometrial side on gd15,and identified expression pattern of them with the technology of immumohistochemical staining and immunofluorescent staining in the paraffin-embedded uterus tissue on ed12,ed15,gd12,gd15.The expression of SPP1?SULT2A1 and THY1 are only existing on the uterus epithelium cells adjacent to the conceptus at mesometrial side on gd15,and the expression of MEP1 B is existing on the uterus epithelium cells on all four stage,except for the uterus epithelium cells adjacent to the conceptus on gd15.For further identification of relationship with embryo specific location implantation,we used paraffin-embedded uterus tissue that conceptus located in the mesometrial side,anti-mesometrial side and between them to identified expression pattern on uterine epithelium cells on gd15.We found the expression of SPP1?SULT2A1 and THY1 are located on the uterine epithelium cells adjacent to the conceptus and the expression of MEP1 B is located on the the uterine epithelium cells away from the conceptus,which show that these genes take part in the process of embryo specific location implantation.(2)Identification of exchange of calcium ion status at estrus and gestation.We found deposit calcium mainly exists on estrus and free calcium exists on gestation using alizarin red S staining with the paraffin-embedded uterus tissue from ed12,ed15,gd12,gd15.And we identified that ANXA8 and S100A9 may be related with calcium status' s change using immumohistochemical staining.The uterine epithelium cells expression of S100A9 on gd12 and gd15 is more intense than that on ed12 and ed15,and the expression of ANXA8 on uterine epithelium cells is only expressed on gd15.The results show S100A9 and ANXA8 may be related with embryo specific location implantation.(3)Acquisition of key transcription factors of uterus luminal epithelium cells during pig embryo implantation.In order to identify transcription factors related with embryo implantation,we obtained uterine epithelium cells at mesometrial side on gd12 and gd15 to do ATAC-seq and analysed the data.We got 789 peaks on gd12 and 488 peaks on gd15.We got 76 transcription factors on gd12 and 90 transcription factors on gd15,such as CEBP?,FOXA1,IRF1,et al.,which may be involved in embryo specific location implantation.4 Analysis of uterine epithelium cells-embryo interaction during pig embryo implantationIn order to study potential interaction relationship between uterine epithelium cells and embryo,we constructed a network website containing uterine epithelium cells-embryo interaction in the maternal-embryo interface with the RNA-seq data of embryo and uterine epithelium cells at mesometrial side and anti-mesometrial side.From the website,we identified some maternal-embryo interaction relationship.For example,SPP1 may be significantly related with HOMER2,SULT1E1,EGR1;SULT2A1 may be significantly related with ZBP1?CLDN23?TSPAN16;THY1 may be significantly related with NFKBIE?SLC7A2?TMEM158;MEP1B may be significantly related with LYPD3?CXCL14?SCNN1D.This network provided a reference for studying interaction between uterine epithelium cells and embryo. |
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