The Role And Signaling Pathway Of IGF-Ⅱ In Embryo Implantation

Embryo implantation is a complex process regulated precisely bymultilevels, such as hormones, growth factors, cytokines and other molecules. Insulin-like growth factors (IGFs) are one of the growth factor family and believed to be important in the cyclic development of endometrium and the embryonic implantation. IGF-II is the main peptide expressed at the maternal-fetal interface in human early pregnancy andlGFBP-1 is the major binding protein of IGFs. The results of indirect immunofluorescence and laser scanning confocal microscopy in this study showed that IGF-II and IGFBP-1 were specifically located at the maternal-fetal interface. In a co-culture system, IGF-II significantly enhanced the attachment and outgrowth of blastocyst on monolayer of uterine epithelial cells, and IGFBP-1 had no effect on the blastocyst attachment, but markedly inhibited the blastocyst outgrowth. The results of zymography in the medium of embryo culture coated with fibronectin showed that IGF-II enhanced the activities of MMP-2 and MMP-9, while IGFBP-1 did not. In conclusion, IGF-II and IGFBP-1 are expressed on EPC (extra placenta cone) during outgrowth phase and regulate blastocyst implantation. IGF-II may regulate blastocyst invasion via MMP-2 and MMP-9, while IGFBP-1 may indirectly inhibit trophoblast invasion by interaction with IGF-II.It is well known that IGF-II may bind to IGFBP-1 that interact by its RGD sequence with integrin a 5 p 1 located on the surface of trophoblast cells, so it is suggestive of paracrine interactions between IGF-II and IGFBP-1 and integrin a 5 3 1 at the maternal-fetal interface. In the current study, the combination of IGF-II and IGFBP-1 did not stimulate the attachment andoutgrowth of blastocyst on monolayer of uterine epithelial cells in a co-culture system, but only IGF-II is able to stimulate the attachment and outgrowth and IGFBP-1 may counteract the effect of IGF-II. The antibody against integrin a 5 p 1 has a marked inhibitory effect on the embryo attachment and outgrowth, but the effect could be counteracted by IGF-II and IGFBP-1. The results of zymography in the medium of embryo culture coated with fibronectin showed that the combination of IGF-II and IGFBP-1 has no obvious effect on the activity of MMP-2 and MMP-9, yet the antibody against integrin a 5 p 1 markedly inhibited the activity of MMP-2 and MMP-9, which can be counteracted by IGF-II and IGFBP-1. Therefore, the equilibrium between the invasion of trophoblast and the inhibition of deciduas may be modulated by the interaction of between the IGF-II and IGFBP-1 on the maternal-fetal interface, and the integrin a 5 P 1 is a modulator of interaction between IGF-II and IGFBP-1.Based on the above results, the next experiment was designed to elucidate the detailed functional role and signaling pathway of IGF-II in embryo implantation. Using JAR cell, a well propagated human first trimester extravillous trophoblast cell line in vitro, we examined the effect of IGF-II on the proliferate and migratory ability and the activity of MMP-2. MTT assay and Blind Well Chamber assay showed that IGF-II enhanced the proliferate and migratory ability and the activity of MMP-2 in JAR cell. To find the detailed signaling pathway, we examined whether the functional role of IGF-II is mediated by G-protein coupled receptor and MAPK pathway. Results showed that IGF-II stimulated the phosphorylation of Erk1/2 at the dose and time dependent manner, reaching the maximum by the concentration of 7 nM at 5 min followed by a decline up to 60 min. The immunofluorescence analysis showed that IGF-II induced the phosphorylation of Erk1/2 that translocated tothe nucleus, but this inducation was blocked by PTX (pertussis toxin), which specially inactivated Gi/o protein. Meanwhile, IGF-II stimulated the phosphorylation of P38 at the time and dose dependent manner, reaching the maximum by the concentration of 10 nM at 5 min. Addition of PTX and PD (a specific MEK inhibitor) and SB ( a specific P38 inhibitor) significantly blocked the proliferation and mig...