Degradation of extracellular matrix by mouse trophoblast outgrowths: a model for implantation

Abstract

During implantation the embryo attaches to the endometrial surface and trophoblast traverses the uterine epithelium, anchoring in the uterine connective tissue. To determine whether trophoblast can facilitate invasion of the uterus by degrading components of normal uterine extracellular matrix, mouse blastocysts were cultured on a radio-labeled extracellular matrix that contained glycoproteins, elastin, and collagen. The embryos attached to the matrix, and trophoblast spread over the surface. Starting on day 5 of culture there was a release of labeled peptides into the medium. The radioactive peptides released from the matrix by the embryos had molecular weights ranging from >25,000 to <200. By day 7 there were areas where individual trophoblast cells had separated from one another, revealing the underlying substratum that was cleared of matrix. When trophoblast cells were lysed with NH4OH on day 8, it was apparent that the area underneath the trophoblast outgrowth had been cleared of matrix. Scanning electron microscopy and time-lapse cinemicrography confirmed that the digestion of matrix was highly localized, taking place only underneath the trophoblast, with no evidence of digestion of the matrix beyond the periphery of the trophoblast outgrowth. The sharp boundaries of degradation observed may be due to localized proteinase secretion by trophoblast, to membrane proteinases on the surface of trophoblast, or to endocytosis. Digestion of the matrix was not dependent on plasminogen, thus ruling out a role for plasminogen activator. Digestion was not inhibited by a variety of hormones and inhibitors, including progesterone, 17fl-estradiol, leupeptin, EDTA, colchicine, NH4CI, or E-aminocaproic acid. This system of culturing embryos on extracellular matrix may be useful in determining the processes that regulate trophoblast migration and invasion into the maternal tissues during implantation.Implantation is an invasive process that is limited in both time and space. In the mouse it begins on day 5 of gestation, with the initial apposition of the embryo to the endometrial surface, and extends to the time that the invading trophoblast taps into the maternal blood vessels. During this period the trophoblast traverses a uterine epithelium composed of cells and connective tissue matrix and penetrates the basement membrane before entering the uterine stroma (26). Although the actual mechanisms have not been elucidated, morpliologic study (26) suggests that entry of trophoblast into the endometrium does not depend on the release of a cytolytic enzyme because maternal cells surrounding the implanting embryo remain largely intact.Attachment of blastocysts onto glass or plastic and subsequent trophoblast outgrowth has been used as a model to study proteinase activity and the interaction of the embryo with other cell types in culture (10,20,30). Studies in culture with timelapse cinemicrography have shown that there is contact inhibition between trophoblast and co-cultured dispersed ceils (10). Trophoblast displaces ...

92‐kDa type IV collagenase and TIMP‐3, but not 72‐kDa type IV collagenase or TIMP‐1 or TIMP‐2, are highly expressed during mouse embryo implantation

“…In fact, these enzymes may not need specific activators as they are autoacti-vated in vitro (Bergmann et al, 1995). Also, €-aminocaproic acid, a n inhibitor of plasminogen activators, does not inhibit rabbit blastocyst attachment or degradation of matrix by mouse trophoblasts in vitro (Denker, 1977;Glass et al, 1983) while inhibitors of metalloproteinases do so (Behrendtsen et al, 1992). It is therefore possible that the plasminogen activatorlplasmin system has an insignificant function in degradation of matrix components but could, alternatively, regulate fibrin formation in the area where maternal blood vessels are breached.…”

Global Ablation of the Mouse Rab11a Gene Impairs Early Embryogenesis and Matrix Metalloproteinase Secretion

“…Previous studies have demonstrated that the degradation of the extracellular matrix by blastocyst outgrowth could be used as an in vitro model for embryo implantation (55,57). To directly assess the in vivo regulation of gelatinase (e.g.…”

Genes for extracellular-matrix-degrading metalloproteinases and their inhibitor, TIMP, are expressed during early mammalian development.

“…1C). There was a large increase in the activity of proteinases, particularly the 92-kD gelatinase, during blastocyst outgrowth, a process corresponding to acquisition of invasiveness during implantation and early postimplantation development in vivo (Glass et al 1983). Cultured egg cylinder-stage embryos secreted even more of these proteinases.…”