The Role Of Autophagy In The Mouse Uterus During Peri-Implamtation Period

ObjectiveEmbryo implantation is a key part of pregnancy,establishing a close relationship between implantable embryos and receptive uterus.Early pregnant mouse uterus relies on the following steps to complete the embryo implantation process: on pregnancy day4,mouse uterus is in the receptive state,allowing the blastocyst adhesion,on pregnancy day5,blastocyst adhere to the endometrium.After the establishment of implantation,endometrial stromal cells begin to under decidualization.On pregnant day6 primary decidualization was established.Successful embryo implantation plays an important role in pregnancy maintenance and childbirth.However,the embryo implantation mechanism is still unclear.Autophagy is a common phenomenon in eukaryotic cells that relies on lysosomes to degrade substances to maintain cell homeostasis and update of intracellular substances.Recent studies have shown that autophagy is involved in the development of tumors.Embryo implantation is similar to the tumor occurrence.But it is still unclear whether autophagy is involved in the embryo implantation process.Pervious study found that the expression of autophagy-related markers was lower in the implantation site of mice on pregnant day5.This suggests that autophagy may be involved in the embryo implantation.This study not only helps to understand the mechanism of pregnancy,but also provide new ideas to women's reproductive disorders.Methods1.Expression of autophagy related marker in endometrium during peri-implantation mice1)The model of pregnant mice was established and the endometrium of pregnant day4(D4),day5(D5)and day6(D6)was collected.Western Blot was used to detect the expression of autophagy-related markers in the endometrium of pregnant mice.2)The mice model of pregnant D5 was established.The endometrium of implantation site(IS)and non-implantation site(IIS)were collected.The autophagy were searched by transmission electron microscopy.Real-time PCR and Western Blot were used to detect the expression of autophagy-related markers on D5 IS and D5 IIS.3)The autophagy inhibitor 3-MA intervened pregnant mice model was established.The endometrium of implantation site(IS)and non-implantation site(IIS)on D5 were collected.After the establishment of the intervened model,the expression of the decidualization related markers were detected by Western Blot.The morphological changes of the uterus were observed by HE staining.2.The role of autophagy in the decidualization.1)The model of artificial induced decidualization was established.Endometrial tissue from induced decidualized side and control side were collected.Real-time PCR and Western Blot were used to detect the expression of autophagy-related markers.In vitro,mouse uterine stromal cells were isolated and induced to decidualized cells.The expression of autophagy-related markers in stromal cells and decidualized cells was examined using Western Blot.2)The autophagy inhibitor 3-MA intervened decidualization induced model was established.After successful establishment of the model,Western Blot was used to detect the expression of decidualization related markers.HE staining was used to observe the morphology of mouse uterus after autophagy was inhibited.After treated with 3-MA,stromal cells were induced to decidualization.Western Blot was used to detect the expression of decidualization related markers.Results1.Expression of autophagy related makers in endometrium during peri-implantation mice1)The expression of LC3-II and ATG5 were significantly lower in the endometrial tissues in mice on pregnant D5 and D6 than that on D4.2)The number of autophagosomes on pregnancy D5 IS was lower than on D5-IIS.3)The expression of HOXA10,ER-? and PR on pregnant D5 were significantly decreased after 3-MA intervention.2.The role of autophagy in the decidualization.1)The expression of autophagy-related markers P62,ATG5,CathepsinB,ULK1 and BECN1 mRNA were significantly decreased in the decidualized endometrium.The expression of autophagy-related protein LC3-II,ATG5 and P62 were significantly down-regulated in the decidualized endometrium.The expression of LC3-II,ATG5 and P62 proteins were significantly lower in deciduaulized cells than those of un-induced stromal cells.2)The robust and wet weight of deciduomas in 3-MA exposed mice significantly decreased compared with the control group.HE staining showed that the boundaries of deciduaulized cells were unclear under the autophagic inhibitor 3-MA intervention.The expression of HOXA10 and PR protein was significantly down-regulated after autophagy was inhibited.The expression of HOXA10 and PR protein were significantly decreased in vitro after induced stromal cell to decidualization.ConclusionThis study suggests that autophagy may be involved in the maintenance of endometrial function,which may be associated with early endometrial decidualization.But the specific mechanism remains to be further studied.