PROTEINASE EXPRESSION IN EARLY MOUSE EMBRYOS IS REGULATED BY LEUKEMIAINHIBITORY FACTOR AND EPIDERMAL GROWTH-FACTOR

Several proteinases from different multigene families have been implic ated in the uterine invasion required for establishment of pregnancy i n some mammals. In this study, the expression of matrix metalloprotein ase gelatinase B (MMP-9), urokinase-type plasminogen activator (uPA) a nd their inhibitors was investigated during early mouse embryo develop ment. Transcripts for tissue inhibitors of metalloproteinases (TIMP-1, -2,-3) and uPA receptor were detected throughout pre- and peri-implant ation development whilst MMP-9 and uPA mRNAs were first detected in pe ri-implantation blastocysts associated with the invasive phase of impl antation. Through use of in situ hybridization, it was shown that MMP- 9 transcripts were strongly expressed in the network of trophoblast gi ant cells at the periphery of implanting 7.5 day embryos and TIMP-3 tr anscripts were strongly expressed in the decidua immediately adjacent to the implanting embryo. uPA transcripts were preferentially expresse d in the ectroplacental cone and its derivatives. Because these protei nases are regulated by growth factors and cytokines in other tissues, the effect of leukaemia inhibitory factor (LIF) and epidermal growth f actor (EGF) on their activity was investigated. Both LIF and EGF, like the proteinases, have been implicated in peri-implantation developmen t. Blastocysts collected on day 4 of pregnancy were cultured 2 days in TCM 199 + 10% fetal bovine serum to allow outgrowth followed by 24 ho ur culture in defined media containing either LIF or EGF. Conditioned media were assayed for uPA activity by a chromogenic assay and MMP act ivity by gelatin zymography. Both LIF and EGF stimulated uPA and MMP-9 activity in blastocyst outgrowths after 3 days of culture (day 7). Pr oteinase 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 pr oduction of both proteinases. These results demonstrate that proteinas e activity in early embryos can be regulated by growth factors and cyt okines during the implantation process and, in particular, they demons trate the possible involvement of LIF in establishment of the correct temporal programme of proteinase expression.