Expression Of GRIM-19 And Its Effects During Mouse Embryo Implantation

Background & AimEmbryo implantation is the basis for the beginning of life, including positioning, adhesion, implantation, whose regulatory mechanisms is complex and sophisticated. GRIM-19 (gene associated with retinoid-interferon-induced mortality-19) is a member of GRIMs family,part of the mitochondrial complex Ⅰ, which play important role in cell apoptosis and immunity. Some studies have shown that GRIM-19 play important part in early embryonic development, but it remains unclear what role it would play in the embryo implantation. This experiment studies the expression and location of GRIM-19 in mouse endometrium in early pregnancyto explore its function in embryo implantation.On this basis, we study the influence of GRIM-19 on cell apoptosis and immunity by experiment in vivo and in vitro to further explore the mechanism acting on embryo implantation.MethodsIn vivo study:We detected the expression of GRIM-19 in the pregnant uterus of mice by Immunohistochemistry. We studied the expression of GRIM-19 in mouse uterus by RT-PCR and Western blot with early pregnancy、hormone treatment and pseudopregnancy models. TUNEL method was used to detect apoptosis in uterine tissues of pregnant mice.In vitro study:An optimized model of R95-2 spheroids and Bewo monolayer co-culture model was employed to study the effects of GRIM-19 in the embryo-endometrial epithelium attachment; Apoptosis was assayed by flow cytometry, Western blotting and real-time PCR was used to detect the mRNA and protein level of STAT3, TNF-α and IL-Ⅱ.Statistical analysis:Data are given as mean ± standard division (SD) of the results from three or four different samples in each item of the experiment. The cells from each sample were separately evaluated. Differences between two groups were measured by the Student’s t-test. A p value less than 0.05 was defined statistically significant difference.Results1. The immunohistochemical positive staining for GRIM-19 was distributed in the luminal epithelium and glandular epithelium in pregnant mice. GRIM-19 decreased to the lowest level at pregnancy day4. The expression of the GRIM-19 protein and mRNA levels reached the bottom on day 4 of pregnant mice, but, no significantly difference in the uterus of pseudopregnant mice on D1 to D6.2.The expression of GRIM-19 inEstrogen-treated group was decreased, butprogestin-treated group showed no significant difference.3. The ratio of apoptotic cells on D4 was significantly lower than the other days (P <0.05).4. Overexpression of GRIM-19 reducedthe adhesion rate of RL95-2-BeWo co-culture spheroid.5.Flow cytometric analysis the showed that overexpression of GRIM-19 increased apoptosis.6. The mRNA and protein levels of STAT3 and IL-11 inGRIM-19-overexpression group were significantly lower than the control. However, the mRNA and protein levels of TNF-a were observed increased. In GRIM-19-silencegroup, the mRNA and protein levels of STAT3 and IL-11 increased, the expression of TNF-a was decreased.ConclusionGRIM-19 plays an important role in embryo implantation. GRIM-19 plays a role in endometrial receptivity, cell proliferation, apoptosis and immune tolerance, interacting with a variety of cytokines, will likely become a new potential target molecule anti-fertility technology.