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Several proteinases from different multigene families have been implicated in the uterine invasion required for establishment of pregnancy in some mammals. In this study, the expression of matrix metalloproteinase gelatinase B (MMP-9), urokinase-type plasminogen activator (uPA) and their inhibitors was investigated during early mouse embryo development. Transcripts for tissue inhibitors of metalloproteinases (TIMP-1,-2,-3) and uPA receptor were detected throughout pre- and peri-implantation development whilst MMP-9 and uPA mRNAs were first detected in peri-implantation blastocysts associated with the invasive phase of implantation. Through use of in situ hybridization, it was shown that MMP-9 transcripts were strongly expressed in the network of trophoblast giant cells at the periphery of implanting 7.5 day embryos and TIMP-3 transcripts were strongly expressed in the decidua immediately adjacent to the implanting embryo. uPA transcripts were preferentially expressed in the ectoplacental cone and its derivatives. Because these proteinases are regulated by growth factors and cytokines in other tissues, the effect of leukaemia inhibitory factor (LIF) and epidermal growth factor (EGF) on their activity was investigated. Both LIF and EGF, like the proteinases, have been implicated in peri-implantation development. Blastocysts collected on day 4 of pregnancy were cultured 2 days in TCM 199 + 10% fetal bovine serum to allow outgrowth followed by 24 hour culture in defined media containing either LIF or EGF. Conditioned media were assayed for uPA activity by a chromogenic assay and MMP activity by gelatin zymography. Both LIF and EGF stimulated uPA and MMP-9 activity in blastocyst outgrowths after 3 days of culture (day 7). Proteinase activity was assayed again at the 5th to 6th day of culture (day 9 to 10). EGF was found to have no effect whereas LIF decreased production of both proteinases. These results demonstrate that proteinase activity in early embryos can be regulated by growth factors and cytokines during the implantation process and, in particular, they demonstrate the possible involvement of LIF in establishment of the correct temporal programme of proteinase expression.