The Mechanism Of SRIF And SST5 Regulating The Receptivity Of Sheep Endometrial Epithelial Cells Through Notch Pathway

Embryo implantation is a key step in mammalian pregnancy.The endometrium undergoes a series of complex dynamic changes and eventually forms a receptive endometrium,and the timely apoptosis of epithelial cells is crucial in this process.SRIF and SST5 have a wide range of inhibitory effects in cells,including inhibition of cell proliferation,migration and hormone secretion.SRIF and SST5 have been confirmed to exist in human endometrium and participate in the regulation of pathological changes in some endometrium.Therefore,it is speculated that SRIF and SST5 play an important role in endometrial function remodeling.The fertility of Xinjiang native sheep is low,and the early embryo implantation disorder is one of the important factors causing this problem.In order to explore the effects of SRIF and SST5 on the process of sheep embryo implantation,this study identified the expression characteristics of SRIF and SST5 in sheep endometrium,established a receptive sheep endometrial epithelial cell model,and exogenously added SRIF-14 and SST5 inhibitor BIM23056 to detect its effect on cell proliferation,migration,apoptosis and receptivity establishment,and preliminarily clarified the function of SRIF and SST5 in sheep endometrial epithelial cells.It provides scientific data for further analysis of the mechanism of endometrial function remodeling during embryo implantation in sheep,and also lays a foundation for further clarifying the mechanism of early embryo implantation disorder in sheep.Objective:（1）The expression levels of SRIF and SST5 in endometrial tissues and endometrial epithelial cells of sheep were determined,and the receptive endometrial epithelial cells were constructed.（2）To investigate the effects of SRIF and SST5 on the proliferation,migration,apoptosis and receptivity of sheep endometrial epithelial cells;（3）To explore the mechanism of SRIF and SST5 affecting the function of endometrial epithelial cells in sheep.Methods:（1）Uterine tissues were collected from non-pregnant and pregnant sheep on day 17 of pregnancy.SRIF and SST5 in endometrial tissues were localized by HE and immunohistochemical staining,and the expression of SRIF and SST5 in tissues was detected by WB.The sheep endometrial epithelial cells were cultured by the method of isolation and purification of sheep endometrial epithelial cells established in our laboratory,and the SRIF and SST5 in the endometrial epithelial cells were localized by immunocytochemistry.Sheep endometrial epithelial cells were treated with E₂,P₄ and IFNT,and the expression of embryonic elongation factor-related genes was detected by RT-q PCR.ELISA method was used to detect the secretion of PGs.The protein levels of PGR,ER,SRIF and SST5 were detected by WB.（2）CCK-8 method was used to screen the concentration of SRIF-14 and SST5 inhibitor BIM23056.The effects of SRIF-14 and BIM23056 on the proliferation of sheep endometrial epithelial cells were detected by CCK-8 method,and the expression of cell cycle-related genes was detected by RT-q PCR method.The effects of SRIF and SST5 on the migration ability of endometrial epithelial cells in sheep were detected by scratch test.The expression of cell tolerance factors was detected by RT-q PCR,and the secretion of PGs was detected by ELISA.PGR and ER protein levels were detected by WB;（3）SRIF-14 and BIM23056 treated sheep endometrial epithelial cells,WB method to detect the protein levels of Caspase 9,Bax,Bcl-2,Cyt-C and Notch;Notch signaling pathway activator（JAG-1）and inhibitor（DAPT）treated sheep endometrial epithelial cells,WB method to detect the protein expression levels of SRIF and SST5,RT-q PCR method to detect the expression of cell tolerance factors,ELISA method to detect the secretion of PGs;WB method to detect the protein levels of PGR and ER.Results:（1）SRIF and SST5 were highly expressed in the endometrium of sheep on day 17 of pregnancy.The receptive endometrial epithelial cells were successfully constructed using E₂,P₄ and IFNT.The m RNA expression levels of proembryonic elongation factor-related genes were significantly increased under the stimulation of E₂,P₄ and IFNT.The secretion level of PGE₂ was significantly increased,the secretion level of PGF_2αwas significantly decreased,and the protein expression level of PGR was significantly decreased.The expression levels of SRIF and SST5 protein in sheep endometrial epithelial cells were significantly increased after E₂,P₄ and IFNT treatment.（2）The drug SRIF-14 and the SST5 inhibitor BIM23056 inhibited cell proliferation in sheep endometrial epithelial cells,while SRIF treatment reduced CCNA expression levels and BIM23056 had no significant effect on cell cycle-related genes;SRIF-14 promoted cell migration and BIM23056 inhibited cell migration.The m RNA expression levels of OPN,ITGB3 and CLDN4 in endometrial epithelial cells of SRIF sheep were increased,the levels of ER protein and PGE₂ secretion were increased,and the levels of PGR protein and PGF_2αsecretion were decreased.After inhibiting SST5,the expression levels of OPN and CLDN4 were significantly reduced,and the secretion levels of PGF_2α,PGR and ER proteins were also significantly decreased.（3）SRIF-14 treatment of sheep endometrial epithelial cells induced increased Caspase 9,Bax and Cyt-C protein expression and decreased Bcl-2 expression levels;after SST5 inhibition,Caspase 9 and Bax protein expression levels were decreased and Bcl-2 expression was increased.Notch signaling pathway was activated after exogenous addition of SRIF,and Notch signaling was also inhibited after inhibition of SST5 expression.Treatment of sheep endometrial epithelial cells with the Notch activator JAG-1 significantly increased the expression levels of SRIF and SST5,increased the expression of cellular tolerance factors OPN and ITGB3,increased the secretion level of PGE₂,and decreased the protein levels of PGR and ER.Notch pathway inhibitor DAPT treatment also inhibited the expression levels of SRIF and SST5,decreased the expression levels of ITGB3 and CLDN4,and decreased the expression levels of ER protein,while the expression levels of PGR and PGs were not affected.Conclusion:In this study,we found that the expression levels of SRIF and SST5 in the endometrial tissue of sheep on the 17 th day of pregnancy were significantly increased.After E₂,P₄ and IFNT were used to stimulate the endometrial epithelial cells of sheep to successfully construct the receptive endometrial epithelial cells,the protein expression levels of SRIF and SST5 in the cells were also significantly increased.Using SRIF-14 and SST5 inhibitors,it was found that exogenous addition of SRIF increased cell migration,apoptosis and tolerance,and induced activation of Notch signaling pathway.After SST5 inhibition,the levels of cell proliferation,migration,apoptosis and tolerance were decreased,and the Notch pathway was also inhibited.The protein expression levels of SRIF and SST5 in sheep endometrial epithelial cells treated with Notch pathway activator were significantly increased,and the cell tolerance level was also increased.After the Notch signaling pathway was inhibited,the protein expression levels of SRIF and SST5 were also inhibited,and the cell tolerance level was reduced.In summary,SRIF and SST5 can regulate the basic functions of sheep endometrial epithelial cells through the Notch signaling pathway and affect the functional remodeling of sheep endometrial epithelial cells,thereby regulating the embryo implantation process.