Embryo implantation in humans and rodents is a highly invasive yet tightly controlled process involving extracellular matrix (ECM) degradation. Matrix metalloproteinase 9 (MMP-9) has been implicated as the major facilitator of this ECM degradation. MMP-9 is expressed by the embryo's trophoblast cells, whereas tissue inhibitor of metalloproteinases 3 (TIMP-3) is expressed by the maternal uterine cells immediately adjacent to the trophoblast. We examined the functional roles of MMP-9 and TIMP-3 during in vitro ECM degradation by mouse embryos. Blastocysts were treated with either MMP-9 antisense or sense oligonucleotides and incubated on an ECM gel. The extent of ECM degradation exhibited by the blastocysts due to proteinase secretion was quantified. Embryos exposed to MMP-9 antisense oligonucleotides exhibited reduced ECM-degrading activity as compared with controls, and this reduced activity was correlated with the level of MMP-9 secreted by the embryos. The functional role of TIMP-3 was then examined by incubating blastocysts on an ECM gel that had been impregnated with various amounts of TIMP-3. In a dose-dependent manner, increases in TIMP-3 resulted in a reduction in ECM degradation and were correlated with diminished MMP-9 activity. These results provide important functional evidence that in vitro ECM degradation is regulated by embryo-derived MMP-9 and ECM-derived TIMP-3.
Several extracellular matrix (ECM)-degrading proteinases are hypothesised to play important roles during early mammalian development. In particular, urokinase-type plasminogen activator (uPA) and matrix metalloproteinase 9 (MMP-9) are expressed in peri-implantation mouse, sheep, and pig embryos and are implicated in the implantation process. These proteinases are not expressed in early (pre-blastocyst) mouse, sheep or pig embryos. The aim of this study was to establish the gene expression and proteolytic activity of uPA and MMP-9 in in vitro-produced (IVP) cow embryos. Using RT-PCR, mRNA transcripts for uPA and MMP-9 were detected during the first 7 days of development. To investigate the activity of these proteinases, conditioned media from various stages of development (days 2, 3, 4, 5 and 7) were assayed for uPA activity by chromogenic assay and MMP-9 activity by gelatin zymography. Both uPA and MMP-9 activities were detected in the media samples indicating the production and secretion of these proteinases. This pattern of proteinase expression is novel in comparison to the mouse where uPA and MMP-9 are only expressed from the blastocyst stage onwards. The results of this study suggest that these ECM proteinases have a role prior to implantation in the cow, in contrast to that exhibited by mouse, sheep and pig embryos.
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