Expression And Regulation Of MLKL Gene In Mouse Uterus During Early Pregnancy

Regular cell death plays an important role both in the entire development process and in maintaining the homeostasis of inner environment.Many studies in recent decades have shown that physiological and pathological necrosis can occur in a regular manner.This regular necrosis is called necroptosis,a type of programmed cell death that is not dependent on the activity of caspase.This process is induced by a combination of biochemical and molecular events at different cellular levels.The most widely studied is TNF-induced programmed cell necrosis,while protein kinase receptor-interacting protein kinase 3（RIPK3）and its downstream substrate:mixed lineage kinase domain-like protein（MLKL）are key cellular components in the downstream signal pathway.Studies have shown that apoptosis and autophagy involved in the process of mammalian embryo implantation,so we have studied whether necroptosis is also important in embryo implantation.To investigate whether necroptosis occurred during the early pregnancy in mice,we studied the expression and regulation of MLKL in mouse early pregnancy uterus and its decidual.We constructed different mouse models:mouse early pregnancy model,artificially induced decidualization model and hormone treatment model;cultured human endometrial stromal cells and induced decidualization through cAMP,MPA,and E₂ in vitro;used real-time PCR,in situ hybridization,western blot and other experimental techniques to analyze the expression patterns of MLKL mRNA and protein in mouse early pregnancy uterus,decidualized uterus,and hormone treatment uterus.In the mouse early pregnancy uterus,before embryo implantation（pregnancy day1-4）,and at the non-implantation site on day 5,the expression of MLKL mRNA and protein was low.But in pregnancy day 5,after entering the embryo implantation process,the expression of MLKL gradually increased and the expression peak was detected in pregnancy day 7,but the expression decreased slightly on day 8 of pregnancy.The results of in situ hybridization showed that contrasting with non-implantation site of day 5,MLKL mRNA was expressed in endometrial luminal epithelium in day 5 of pregnancy.And day 6-8 of pregnancy was mainly expressed in decidua.In the artificially induced decidualization model uterus,the expression of MLKL was very high,while not significantly expressed in the control groups.And in Induced human decidualized cells,the expression of MLKL mRNA gradually increased with the induction time.In the hormone-treated model uterus,the MLKL mRNA and protein expression levels were significantly higher in the uterus of the treatment group injected with P₄ than in control group（P<0.05）.In the uterus of the group treated with E₂ alone and E₂ and P₄ together,the expression of MLKL was was similar to the control group.The experimental results show that MLKL is closely related to embryo implantation and decidualization in mice,which also indicates that necroptosis plays a role in the pregnancy process of mice.And MLKL expression is regulated by steroid hormones,especially progesterone.The highly expressed MLKL gene in mouse uterine decidua may regulate the differentiation of uterine stromal cells.These results demonstrate that MLKL plays an important role in mouse embryo implantation and decidualization.