Study On The Function And Mechanism Of IFN-? And Biomechanical Activating YAP To Maintain Reproductive Ability Of Dairy Cows

Subfertility and infertility are pervasive issues that are common in the field of mammalian reproduction.As one of the main organs of the reproductive system and an important site for the growth and development of the fetus,the embryonic death and pathological damage caused by its dysfunction during pregnancy were key factors restricting maternal fertility.Mammalian tissues and organs,including the uterus of cows,are stimulated by both biochemical and biomechanical signals from their surroundings.The current research on uterine function focuses on the regulation of the uterus by soluble chemical signals such as steroid hormones.Previous studies have shown that biomechanical were the main driving force for morphogenesis,definition of tissue structure and maintenance of tissue homeostasis during embryonic development.However,there are few studies on biomechanical that regulate uterine fertility in dairy cows.This study systematically analyzed the mechanism of biochemical signals in the uterine environment that cooperate with biomechanical to maintain early pregnancy and repair after injury in dairy cows on the overall level,expanding the understanding of female animal fertility,and helping to improve the actual problems of dairy cow production,which then improving the efficiency of breeding.In addition,uncovering the repair mechanism of the uterus also has great significance for the field of regenerative medicine.1.Firstly,we conducted a secondary excavation of the endometrial RNA-seq data(GSE107891,GSE107891,and GSE46274)of early pregnancy and unpregnant cows in the GEO database.GO analysis found that the extracellular matrix(ECM)components and cell-cell,Cell-ECM adhesion had changed significantly,which implied that the biomechanics in the uterus had changed.Next,this result was verified by immunofluorescence(IF)technology and Western blotting(WB)in collecting cow uterus samples.And it was found that YAP(Yes-associated protein)in the endometrium of pregnancy had significantly high expression at the protein level and gene level.IF and immunohistochemistry(IHC)experiments observed the subcellular localization of YAP and found that YAP in pregnant uterus had nuclear translocation,and it was most obvious in cow endometrial epithelial cells(b EECs).2.In order to reveal the molecular mechanism of biomechanical mediation pregnancy,this study successfully established a density gradient model and an ECM stiffness model.b EECs were first cultured with low density(< 40%),high density(> 80%),soft matrices(1 k Pa)and stiff hydrogels(40 k Pa),then conducted the nucleoplasmic protein separation.Low density and 40 k Pa induced a significant increase in YAP expression and subcellular localization of nuclear metastasis,which meant inducing YAP activation.After knockdown LATS 1/2,which is upstream of the classical Hippo-YAP pathway,the low-density-induced YAP activation was abolished,but the activation of YAP with 40 k Pa could not be blocked.This indicated that the ECM stiffness transduction was independent of the Hippo pathway and the cell density transduction depends on the Hippo pathway.Further study,Lat.A,an inhibitor of F-actin polymerization,could block the activation of YAP by cell density and ECM stiffness,which indicated that the above-mentioned biomechanical transduction required F-actin expression and polymerization.IFN-? is a unique pregnancy recognition signal during early pregnancy of ruminants.This study speculated that IFN-? was a special biochemical cue involved in the regulation of YAP activity.The results confirmed that YAP could be activated by IFN-? in a concentration-dependent manner and does not affect the phosphorylation level of YAP.Based on the previous mi RNA sequencing data of IFN-? stimulated b EECs by our lab,combined with the prediction results of mi RNA which targeted YAP by bioinformatics software,it was screened out that mi R-16 a might be involved in IFN-?-induced YAP activation.The dual luciferase report experiment confirmed that YAP was the direct target of mi R-16 a.In vitro,transfection of mi R-16 a mimics/inhibitors could interfere with IFN-?-induced YAP activation,revealing the biochemical signaling mechanism of IFN-? /mi R-16 a / YAP in b EECs.3.When using ECM stiffness,IFN-?,or si YAP and overexpression plasmids to alter the expression of YAP in vitro,it could be seen that YAP activation induced the proliferation of b EECs and promotes the EMT process and weak inflammatory environment during early pregnancy.Based on the above results,this study uses mice as model animals to establish normal pregnancy models,pseudopregnancy models,and IFN-? alone treatment models.Based on WB,IF and other experiments,the activation of YAP induced by biomechanical and biochemical signal in early pregnancy is verified in vivo.In addition,intraperitoneal injection of YAP inhibitor Verteporfin(VP)or intrauterine injection of si YAP inhibited the proliferation of endometrial cells,reducing the number of embryos and thereby destroying the establishment of early pregnancy in mice.4.Rapid repair after uterine injury is the most critical factor in maintaining the reproductive capacity of dairy cows and shortening the calving interval.In order to explore the role of the above-mentioned biomechanical in repairing the uterus after injury,this study established mouse uterine injury models.According to the morphological indicators,histopathological scores,molecular repair markers and other standards during the repair period,the repair time of the two models was defined for the next step.The results showed that in the 3×LPS mice model(3 consecutive intrauterine injections with intervals of 7 days),the 18 th day was the beginning of the repair,the 20 th day being the peak of the repair,and the 22 nd day of the repair basically completed.The corresponding postpartum repair time is 0 d,5 d,and 10 d.In order to explore whether biomechanical are involved in repair after injury,this study integrated public RNA-seq data(GSE111976 and GSE40312),and Gene Set Enrichment Analysis(GSEA)showed that cell-cell and cell-ECM adhesion during the repair phase,and ECM components regulate abnormal changes in genes.IF detected epithelial polarity indexes ?PKC and E-cadherin,cell connection indexes ZO-1 and F-actin,and together with the data change in area after 24 h of uterine whole tissue cultured in vitro.It was concluded that the biomechanical was abnormal in the injury-repair period.Further research found that YAP showed a trend of activation during the repair phase,and after using VP and si RNA to interfere with YAP activation,the repair time of uterus was significantly prolonged.5.In order to explore the mechanism of mechanotransduction during the repair phase,this study isolated endometrial stromal cells(ESCs)as an in vitro carrier.Different from previous studies,this study found that Rap1 a mediates the regulation of ECM stiffness on YAP activation,and that the Rho GAPs family member ARHGAP35 combined with Rap1 a in ESCs cells seeded on 1 k Pa hydrogel.Knockdown of ARHGAP35 expression by si RNA could destroy YAP cytoplasmic retention caused by low ECM stiffness or overexpression of Rap1 a.Further studies showed that Rho A participating in the mechanotransduction mediated by ARHGAP35.The Rho A inhibitor C3 and Y27632 as well as the F-actin inhibitor Lat.A and cytochalasin B(Cyto.B)were used both in vivo and in vitro experiments.The results revealed a new biomechanical transduction mechanism,namely Rap1 a / ARHGAP35 / Rho A / F-actin / YAP,interfering with the conduction of this pathway and directly extending the repair period after uterine injury.Conclusion:YAP in endometrial cells responds to the conduction of biomechanical and chemical signals participate in the maintenance of uterine fertility through regulating the repair process after uterine injury and the establishment of early pregnancy.