| The fallopian tube mucosa maintains the internal environment for gamete transport and embryo development in the fallopian tube lumen.Mucosal damage and immune dysfunction caused by infection or chronic inflammation can lead to diseases and reduce the production capability of female animal,estrogen has crucial regulatory effects on the immune balance of the fallopian tube mucosa.In this study,transcriptome analysis was used to find the key factors of estrogen regulating the immune function of oviduct mucosa,and further to explore the signaling pathway it is modulated and immune regulation function.This study supplements the molecular mechanism of estrogen regulating in oviduct mucosal immunity,and has a guiding role in the recovery of inflammatory damage of the oviduct.The research content is as follows:RNA-seq was used to detect the differentially expressed genes(DEGs)of oviduct epithelial cells(OECs)after 10-8 M E2 treatment for 0,1.5,3.5 and 5.5 hours.The results showed that significantly changed DEGs in different time groups changed dynamically,with a maximum of 324 DEGs.GO function enrichment analysis showed that,the treatment of E2,it affected the immune function,cell proliferation and division,cell differentiation,cell connection,nutrient metabolism and other activities of the oviduct mucosa,the DEGs enriched in immune-related functions include S100A8,S100A9,S100A12,PTX3,LBP,NUPR1,CXCR4,etc.We found for the first time that E2 had a significant dynamic regulation effect on immunoregulatory factors S100A8,S100A9,and S100A12 in OECs,the changes of S100A8,S100A9 and CXCR4 after the use of E2 was significantly correlated.KEGG analysis found that DEGs have been enriched in cytokine signaling pathway,arachidonic acid metabolism,MAPK signaling pathway,leukocyte transendothelial migration,calcium signaling and other pathways.Tested by immunohistochemistry method,S100A8 and S100A9 are abundantly expressed in the oviduct mucosal epithelium of healthy sheep in interestrus.10-8 M E2 were used on OECs at different time(5h,6h,7h,8h),q-PCR showed that S100A8 and S100A9 were significantly higher than the control group after 7 hours of exposure and reached a peak value.All of the different consentration(10-5 M,10-6 M,10-7 M,10-8 M,10-9 M)of E2 up-regulated S100A8 and S100A9,the expression levels of S100A8 and S100A9 in the 10-7 M and 10-8 M groups were the highest,but the difference between the two groups was not significant.Immunocytochemistry experiments also showed that the expression of S100A8 and S100A9 increased after 7 hours of exposure to 10-8 M E2.After treatments of three estrogen receptor inhibitors,q-PCR and WB method were used to detect the changes of S100A8 and S100A9 in OECs.The results showed that either ER inhibitors Tamoxifen,Fulvestrant or GPER antagonists G-15 inhibited the expression of S100A8 and S100A9,so E2 activates both the classical receptor ER and the membrane receptor GPER,and up-regulates the expression of target genes S100A8 and S100A9 together.After treatments of three MAPK kinase family inhibitors,q-PCR and WB method were used to detect the changes of S100A8 and S100A9 in OECs.It was found that the JNK inhibitor SP600125 had a significant suppression on the expression of S100A8,while the P38 MAPK inhibitor SB203580 and ERK1/2 inhibitor U0126 were found no suppression on S100A8 in mRNA and protein level.Effects of several estrogen receptor inhibitors on activation of JNK after the E2 treatment in OECs,which showed that ER inhibitors Tamoxifen,Fulvestrant and GPER inhibitors G-15 inhibited the activation of JNK,indicating that E2 activate JNK through different membrane estrogen receptor ER and GPER.In summary,E2 could activate JNK through different mER and affect the expression of S100A8.Two-gRNA CRISPR/Cas9 method are used to knock down the S100A8 gene in oviduct mucosal epithelial cells,and detect the changes of related immune factors IL-1? and IL-10.The results showed that the knockdown of S100A8 will lead to the decrease of IL-10 and the increase of IL-1?,indicated that S100A8 upregulates the anti-dinflammatory factor IL-10 and downregulates the inflammatory factor IL-1? at certain extent under physiological conditions,which maintains the immune homeostasis of the fallopian tube.In addition,S100A8 may regulate the immune tolerance by IL-10 at high concentration of estrogen in the fallopian tube.When the S100A8 gene was knocked down,IL-10 is upregulated by estrogen,this may be the compensating mechanism. |
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