The Local Regulation Mechanism Of Glucocorticoid Balance In Mouse Endometrium

Embryo implantation and decidualization are two key processes during the mouse early pregnancy.The failure in any step can lead to infertility,miscarriage and other adverse outcomes.Stress can increase glucocorticoid level in the body,thus causing many reproductive diseases,such as premature labor,fetal growth restriction,abortion and fetal chronic diseases.Although there are many studies on the adverse effects of excessive glucocorticoid on pregnancy,there are few studies on the effects of glucocorticoid on embryo implantation and decidualization,especially on the underlying mechanisms.Hsd11b2 can inactivate the glucocorticoid,and is highly expressed in human and mouse placenta,which establishes a glucocorticoid barrier in placenta.The placental glucocorticoid barrier can protect the fetus from the influence of maternal glucocorticoid level to a certain extent.However,it is not known whether a glucocorticoid barrier exists in the uterus before placentation.Therefore,in situ hybridization,Western Blot,q PCR,immunofluorescence and other techniques were used to explore the expression and regulation of Hsd11b2 in mouse uterus during the early pregnancy,and the effects of stress on embryo implantation and decidualization.From days 1-8 of mouse pregnancy,Hsd11b1 expression was mainly located in stromal cells in the secondary decidual area of the uterus.Hsd11b2 signal was located in stromal cells at the anti-mesometrial side on days 3-4 of pregnancy,and in stromal cells at implantation sites on day 5.Results of q PCR showed that the m RNA expression level of Hsd11b1 was gradually down-regulated from days 1-4 of pregnancy,while the expression level of Hsd11b2 was the opposite.The protein level of Hsd11b2 was also significantly up-regulated on days 3-4 of pregnancy.The m RNA expression level of Hsd11b1 in mouse uterus was down-regulated by progesterone in ovariectomized mice,while Hsd11b2 was up-regulated.When mice were injected with RU486 on day 3 of pregnancy,the m RNA expression level of Hsd11b2 was significantly down-regulated.The m RNA expression level of Hsd11b2 in stromal cells was significantly up-regulated by progesterone,which was abrogated by RU486.These results indicate that progesterone can directly regulate the expression of Hsd11b2 through PR.Both the m RNA and protein expression level of Hsd11b2 were obviously up-regulated after stromal cells were treated with purmorphamine,an agonist of IHH signaling.The m RNA expression level of Hsd11b2 was down-regulated after stromal cells were treated with cyclopamine,an antagonist of IHH signaling.The m RNA expression level of Hsd11b2 was also down-regulated after stromal cells were treated with GANT61,an antagonist of IHH signaling.These results indicated that Hsd11b2 is indirectly regulated by the IHH signaling pathway.The enzymatic activity assay of stromal cells showed that progesterone increased the enzyme activity of Hsd11b2.The increased serum concentration of corticosterone in the stress-treated mice verified the effectiveness of SPS and corticosterone injection.When the ovariectomized mice were treated with SPS,the m RNA expression level of uterine Hsd11b2 was up-regulated.When both ovariectomized and adrenalectomized mice were treated with corticosterone,the m RNA expression level of uterine Hsd11b2 was also up-regulated.The m RNA expression level of Hsd11b2 was up-regulated after stromal cells were treated with corticosterone.These results indicated that stress could promote the uterine expression of Hsd11b2.When SPS or corticosterone injection was performed every morning from days 1-4 of pregnancy,the embryo implantation rates decreased on day 5.After the blastocysts from normal mice were transferred into the uterine lumen of the day 4 of pseudopregnant mice under SPS from days 1 to 3,the rates of embryo implantation on day 6 were significantly reduced.The effect of corticosterone injection on delayed and activated model showed that corticosteron injection inhibited embryo implantation.When SPS or corticosterone injection was performed every morning from days 5-7 of pregnancy,the weight of the implantation sites were reduced on the day 8 of pregnancy.The effect of corticosterone on artificial decidualization model in vivo showed that the weight of deciduoma was significantly reduced.Corticosterone inhibited the expression of Cox-2,a early decidualization marker.The above results showed that the high corticosterone level reduced the implantation rate and inhibited decidualization.After 2-cell mouse embryos were treated with 100 μM corticosterone,the rate of blastocyst formation and hatching,and Oct4 expression were reduced.When SPS or corticosterone injection was performed every morning from days 1-4 of pregnancy,uterine collagen III and collagen IV contents increased on day 4.Collagen III and collagen IV were also significantly increased after stromal cells were treated with corticosterone.When SPS or corticosterone injection was performed every morning from days 5-7 of pregnancy,the number of uterine natural killer cells was decreased on the day 8 of pregnancy.The m RNA expression level of IL15 was significantly down-regulated after stromal cells were treated with corticosterone.These results suggested that a high level of corticosterone inhibited embryo implantation by promoting the accumulation of uterine collagens,and inhibiting Cox-2 expression and the u NK recruitment.In summary,Hsd11b2,as a uterine glucocorticoid barrier,plays an important role in maintaining the uterine glucocorticoid balance and protecting embryo implantation and early decidualization.