早孕小鼠子宫内膜上皮葡萄糖转运体3表达对胚胎着床的作用

Effect of glucose transporter 3 expression in endometrial epithelium on embryo implantation in early pregnant mice

目的探讨早孕小鼠子宫内膜上皮葡萄糖转运体3(GLUT3)的表达对胚胎着床的影响。方法(1)免疫组化及Western-blot法检测孕2 d、孕4 d和孕6 d小鼠子宫内膜上皮GLUT3表达情况。(2)采用RNA干扰技术低表达GLUT3,研究其对胚胎着床的影响:取孕2 d小鼠,一侧子宫角注射25μl特异性GLUT3-siRNA以低表达上皮GLUT3,另一侧子宫角注射等量无义siRNA为对照;然后于孕4 d冲洗小鼠宫腔收集囊胚,同时收集子宫组织,采用免疫组化、免疫荧光和Western-blot法检测孕4 d小鼠子宫内膜接受态标志分子白血病抑制因子(LIF)和整合素ανβ3蛋白的表达,孕6 d检测胚胎着床情况。结果(1)免疫组化结果显示,GLUT3主要在子宫内膜上皮表达,孕2 d上皮GLUT3表达呈强阳性,而基质表达弱;孕4 d和孕6 d上皮和基质都有表达。Western-blot结果显示,随着孕天数增加GLUT3蛋白表达水平也随之升高(P<0.05),且孕6 d着床区GLUT3表达显著高于非着床区(P<0.01)。(2)孕2 d宫腔内注射GLUT3-siRNA低表达上皮GLUT3后,未显著影响小鼠孕4 d囊胚发育(P>0.05),但孕4 d子宫内膜接受态标志分子LIF和整合素ανβ3蛋白表达显著下降(P<0.05),孕6 d胚胎着床数也显著下降(P<0.001)。结论低表达上皮GLUT3使子宫内膜接受态受损,进而影响胚胎着床。

Objective:To investigate the effect of glucose transporter 3(GLUT3)expression in endometrial epithelium on embryo implantation in early pregnant mice.Methods:The expression of GLUT3 in mouse endometrial epithelium on gestational day 2,4,6 was examined by immunohistochemistry and Western blot.Knockdown GLUT3 expression by RNA interference was used to study its effect on embryo implantation.On gestational day 2,25μl special GLUT3-siRNA was injected into one side of the cornua uteri to knockdown GLUT3 expression,while nonsense siRNA was injected into another side of the cornua uteri as control.Then,the mouse uterine cavity was flushed to collect blastocysts,while uterine tissue was also collected on gestational day 4.The expressions of endometrial receptivity marker molecules,i.e.leukocyte inhibitory factor(LIF)and integrinανβ3,were examined by immunohistochemistry,immunofluorescence and Western blot.Implantation sites were observed on the gestational day 6.Results:The immunohistochemical results showed that GLUT3 was mainly expressed in the endometrial epithelium.The expression of GLUT3 in the epithelium was strongly positive on the gestational day 2,while the expression was weak in the stroma.Expression was observed in both epithelium and stroma on the gestational day 4 and 6.The Western blot results showed that the expression level of GLUT3 protein also increased with the increase of gestational days(P<0.05),and the expression of GLUT3 in the implantation area was significantly higher than that in the non-implantation area on the gestational day 6(P<0.01).After intrauterine injection of GLUT3 siRNA to knockdown GLUT3 expression in endometrial epithelial on the gestational day 2,early embryonic development was not affected(P>0.05),but the endometrial expression of LIF and integrinανβ3 were decreased on the gestational day 4(P<0.05),and the number of implanted embryos on the gestational day 6 was also significantly decreased(P<0.001).Conclusions:Knockdown of GLUT3 expression impairs the mouse endometrial receptivity,and further influences the embryo implantation.