Regulating effect of LIF on the expression of FuT7: Probe into the mechanism of sLex in implantation
Regulating effect of LIF on the expression of FuT7: Probe into the mechanism of sLex in implantation
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DOI：
10.1002/mrd.21055
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发表时间：
2009-08
影响因子：
2.5
通讯作者：
Q. Zhang;S. Liu;Zm Zhu;Q. Yan
中科院分区：
文献类型：
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作者：
Q. Zhang;S. Liu;Zm Zhu;Q. Yan
着床是通过各种着床相关因素的相互作用来调节的发育过程(Paria等人，2002年)。许多研究关注着床期在胚胎和子宫中高表达的岩藻糖化低聚糖的功能。子宫中唾液酸化路易斯X(sialyl Lewis X，SLE)和胚泡中L-选择素的表达显著增加，这与以下观察结果一致：在着床窗期，SLE是滋养层细胞识别和黏附子宫内膜的关键介质(Genbacev等人，2003年)。然而，SLE表达的调控及其在植入过程中的作用尚不清楚。本研究的目的是通过SLE岩藻糖基化关键酶FuT7的表达，探讨人类和啮齿动物胚胎着床所必需的细胞因子白血病抑制因子(LIF)与SLE合成的关系。交配后第4天从子宫中采集桑椹胚期小鼠胚胎。在含LIF(0.1、1、10、100 ng/ml)或抗LIF抗体(3 mg/ml)的Ham‘s F-10培养液中分别孵育8h或4h后，用实时定量聚合酶链式反应(Real-time PCR)、直接免疫荧光法和斑点免疫印迹法检测FuT7mRNA和蛋白的表达。给出了三个独立实验的均方差结果。组间差异的统计学处理采用非参数检验，P<；0.05有统计学意义。结果表明，LIF可上调FuT7基因和酶在胚胎中的表达，而抗LIF抗体则相反。在0.1~100 ng/ml浓度范围内，LIF对FuT7基因表达的影响呈剂量依赖性。每毫升LIF的最佳浓度为10纳克，在100 ng/ml时表达减弱，这与LIF浓度过高(100 ng/ml)可能抑制胚胎生长或对胚胎有毒的报道一致。这些结果表明，LIF具有调节胚胎发育的双峰作用，因此在实验和临床应用中应慎重选择LIF的剂量。此外，斑点免疫印迹法检测了LIF对SLE表达的影响，其变化趋势与LIF对FuT7表达的影响趋势一致(图1)。成功的植入需要有能力的胚胎和接受能力的子宫之间的相互对话。SLE在胚胎上的阶段性表达与其识别和黏附能力密切相关。因此，LIF上调SLE的表达可能是LIF促进胚胎发育、提高小鼠胚胎着床率的重要途径(Mitchell等，2002)。总之，我们提供了LIF上调胚胎FuT7基因和蛋白表达的证据。LIF可能通过FuT7影响SLE的表达，从而使SLE与植入调控网络相连。为了阐明这一调控网络的作用，还需要进一步研究其确切的分子机制。
Implantation is a developmental process regulated through the interaction of various implantation-related factors (Paria et al., 2002). Many studies have paid attention to the functions of fucosylated oligosaccharides highly expressed by the embryo and uterus during the implantation period. A marked increase in the expression of sialyl Lewis X (sLe) by the uterus and L-selectin by the blastocyst have been reported, consistent with the observation that sLe are crucial mediators in the recognition and adhesion of trophoblasts to uterine endometrium at the implantation window (Genbacev et al., 2003). However the regulation of sLe expression and its role during implantation is unclear. The objective of our study was to investigate the relationship between leukemia inhibitory factor (LIF), an essential cytokine for embryo implantation in human and rodent, and the synthesis of sLe via the expression of fucosyltransferase VII (FuT7), a key enzyme of sLe fucosylation. The mouse embryos of morula stage were collected at the fourth day after mating from uteri. After incubating morulae in media containing LIF (0.1, 1, 10, 100 ng/ml) or anti-LIF antibody (3mg/ml) contained in Ham’s F-10 medium for 8 h or 4 h, respectively, FuT7 mRNA and protein levels were examined using Real-time PCR, direct immunofluorescence and dot immunoblot. Results are given as means SD for three independent experiments. The statistical significance of differences between groups was analyzed by nonparametric test; P<0.05 was considered to be significant. Our results showed that LIF up-regulated FuT7 gene and enzyme expression in the embryo, whereas anti-LIF antibody had a reverse effect. The effect of LIF on FuT7 gene expression was dose-dependent in 0.1–100 ng/ml concentrations. Ten nanograms per milliliter was optimum and the expression was weakened in 100 ng/ml, which was coincident with the report that the excessive concentration of LIF (100 ng/ml) may restrain growth of the embryo or be poisonous to it. These results demonstrated that LIF has a bimodal function in regulating the development of embryo, hence we must choose the doses of LIF carefully in the experiments and clinical applications. In addition, the effect of LIF on the expression of sLe was also assayed by dot immunoblot, which showed the same tendency as that of LIF on FuT7 expression (Fig. 1). Successful implantation requires cross-talking between the competent embryo and receptive uterus. The stagespecifically expressed sLe on the embryo is closely related to the recognition and adhesion potential. Therefore, increasing the expression of sLe by LIF may be an important way for LIF to accomplish its functions in promoting embryo development and increasing the implantation rate of mouse embryo (Mitchell et al., 2002). In conclusion, we provide the evidence that LIF can up-regulate the gene and protein expression of FuT7 on the embryo. sLe expression may be affected by LIF via FuT7, thus sLe connects with the regulatory network of implantation. To clarify the roles of this regulatory network, further studies on the precise molecular mechanisms are needed.