Expression And Regulation Of PAD4 During Embryo Implantation And Decidualization

ObjectiveThe destruction of endometrium receptivity is the main cause of infertility caused by endometriosis and also an important factor limiting the success of assisted reproductive technology.Peptidyl arginine deiminase 4(PAD4)plays a variety of biological functions in embryonic development,immunity,inflammation and tumorigenesis.The role of PAD4 in embryo implantation and decidualization has not been reported so far.To explore the expression of PAD4 in human and mouse endometrial tissues and its role in endometrial acceptance and decidualization will provide a theoretical basis for assisted reproductive technology.MethodThis topic mainly pregnancy which CD1 mice model was established,false pregnancy,steroid hormone treatment model and induce stromal cells in vitro decidual model and application model of endometrium by detecting PAD4 protein and PAD4 mRNA expression in the endometrium of mice and humans,and then discusses PAD4 in the process of embryo implantation and decidual expression.ResultsImmunofluorescence was used to detect the expression of PAD4 protein in decidualization model of human endometrial stromal cells.PAD4 protein was expressed in both cytoplasm and nucleus of decidualized cells.With the development of decidualization,the expression of PAD4 protein increased at 0h,48h and 96h cytoplasm,but decreased at 144h cytoplasm.The expression of PAD4 protein in the nucleus decreased with decidualization.Immunohistochemistry was used to detect the expression of PAD4 in normal and endometriosis human endometriosis tissues.It was found that the expression of PAD4 protein was stronger in the epithelium of normal endometriosis tissues during the middle secretory period,but weaker in endometriosis tissues.The expression was stronger in normal uterine tissue medium secretory period and endometriosis stromal cells.The expression of stromal cells was weak in normal intimal tissue at late stage.In siRNA knockdown experiment,the mRNA expression of PAD4 was decreased after PAD4 knockdown,and the mRNA expression of IGFBP1 and PRL was increased after decidualization.Compared with normal decidualization group,PRL mRNA expression in PAD4 knockdown group was significantly decreased.The mRNA expression of endometrium PAD4 decreased after knockdown of endometrium PAD4 in endometrium.After decidualization,the expression levels of IGFBP1 mRNA and PRL mRNA in endometrium were increased in endometrium group.Compared with normal group,the expression levels of PRL mRNA and IGFBP1 mRNA in endometrium PAD4 knockdown group were significantly decreased.Real-time PCR was used to detect the mRNA expression of PAD4 in the early pregnancy model of mice.PAD4 mRNA increased from the 0.5 day of pregnancy(D0.5)to the 1.5 day of pregnancy(D4.5),and the expression of PAD4 mRNA decreased gradually with the progress of pregnancy after implantation of D2.5 embryos.On days D4.5 and D5.5,expression was decreased at non-implantation sites compared with implantation sites.In order to investigate whether the expression of PAD4 is related to the existence of embryos,a pseudo-pregnancy model was used to detect the expression of PAD4 mRNA in the uterus of mice.From PD0.5 day to PD5.5 day,PAD4 mRNA expression decreased with gestation progression.The estrogen progesterone regulation model was used to detect the effect of estrogen progesterone regulation on PAD4 mRNA expression in mouse uterine tissue.Real-time PCR showed that the expression of PAD4 mRNA in mice treated with P4 decreased after 6h compared with that in mice without P4 treatment.The mRNA expression of PAD4 in ovariectomized mice treated with E2 alone decreased significantly after 6h treatment.The expression of estrogen combined with progesterone decreased after treatment.To observe the regulation of estrogen progesterone on PAD4 mRNA expression in ovariectomized female mice treated with estrogen progesterone combined inhibitor.Compared with the control group,the expression of PAD4 mRNA was increased in mice treated with P4 alone for 6h,and decreased in mice treated with P4 combined with RU489 for 6h.With subcutaneous injection of sesame oil for 6h as control,the mRNA expression of PAD4 decreased after E2 treatment alone for 6h.The mRNA expression of PAD4 after 6h treatment with E2 combined with ICI decreased compared with that without ICI.Real-time PCR was used to detect the expression of PAD4 mRNA in the decidualization process of mouse stromal cells induced by estrogen progesterone.The expression of PAD4 mRNA was increased at 48 h,72h,96h and 120h after estrogen progesterone-induced decidualization,and decreased at 24h.ConclusionsKnockdown of PAD4 gene can decrease the expression of PAD4 in decidualization,suggesting that PAD4 is involved in decidualization and has a dose-dependent effect on decidualization.Knockdown of PAD4 gene can decrease the expression of PAD4 in endometriosis decidualization,suggesting that PAD4 is involved in endometriosis decidualization and has a dose-dependent effect on endometriosis decidualization.Estrogen and progesterone down-regulated the expression of PAD4 mainly through the estrogen progesterone receptor.The existence of PAD4 is related to embryo implantation in mice,and its effect on mouse uterus is related to the existence of embryo.PAD4 was involved in the decidualization of mouse stromal cells in vitro.