The Role And Mechanism Of USP18 In Goat Embryo Implantation

Low fertility rate has became the primary problem of ruminant reproduction.Embryo implantation is an important step in ruminant pregnancy,and fully revealing the regulation mechanism of embryo implantation has always been the focus of animal reproductive biology.Interferon-τ,specific signal for pregnancy recognition,secreted by ruminant embryos and induces a series of IFN-stimulated genes which plays a key role in inhibiting luteinolysis and promoting embryo implantation by activating the JAK/STATs signal.ISGylation system which is mediated by ISG15 has became important targets for the regulation of embryo implantation.Ubiquitin specific Peptidase 18（USP18）specifically deubiquitinats the ISGylation system and regulates type I IFN signaling.However,its role and mechanism in embryo implantation of ruminants are still unknown.In this study,the early endometrial tissues of goats were collected,and the expression of USP18 during embryo implantation was detected by IHC and q RT-PCR.The effects of P₄,E₂and IFN-τon USP18 were analyzed by immortem goat endometrial epithelial cells（g EECs）in vitro.To clarify the mechanism of USP18 in embryo implantation was by lentivirus interference and overexpression techniques.The main results are as follows:（1）USP18 was mainly located in luminal and glandular epithelial cells in endometrial tissues of goats at 5,15 and 18 days of early pregnancy,and the m RNA expression level of USP18 increased significantly with pregnancy days.（2）The mRNA expression of USP18 was significantly increased after treating with P₄,E₂and IFN-τin g EECs,and it was mainly induced by IFN-τ.And the m RNA expression of UBE1L,UBE2L6,and HERC5 that associated with the ISGylation modification system increased after treating with P₄,E₂and IFN-τ;P₄,E₂and IFN-τin g EECs activated the erpression of of JAK/STAT pathway of relating genes STAT1,STAT2 and IRF9 and phosphorylation of STAT1 protein;（3）The overexpression of USP18 promoted the mRNA expression of marker genes HOXA11 of endometrial receptivity and cell adhesion genes including ITGB1,ITGB3,ITGB5,and significantly decreased the mRNA expression of of epithelial cell marker factor E-cadherin.Interference with USP18 reduced the expression of markers HOAX10 of endometrium receptivity,inhibited the expression of cell adhesion genes including ITGB1,ITGB3 and ITGB5.（4）USP18 inhibited the expression of the ISGylation binding protein and the phosphorylation of p-STAT1 after the g EECs were induced by IFN-τ.（5）Fludara which is the inhibitor of STAT1 activation can counteract the inhibitory effect of USP18 on endometrium receptivity and its role in promoting ISGylation of binding proteins in interference USP18 of gEECs.In conclusion,the expression of USP18 increases in endometrial tissue during early embryo implantation.And USP18 regulates JAK/STAT1 pathway and the process of ISGylation to promote endometrial receptivity.