Immunosuppressive properties of murine trophoblast

The modification of the immunological response by murine trophoblast cells of different sources was investigated using the mixed lymphocyte reaction (MLR) and the cell mediated lympholysis (CML) test. MLR between C57BL (H-2b) stimulator splenocytes (mitomycin C treated in the unidirectional MLR) and BALB/c (H-2d) responder lymph node cells were markedly suppressed by trophoblast of ectoplacental cone (EPC) and placental origin. The same in vitro effect was observed with supernatants (SN) of trophoblast cells and with supernatants of blastocysts. Addition of anti-progesterone serum (APS), anti-testosterone serum (RAT), and anti-immunoglobulin serum (RAHIg) in serial dilutions to the trophoblast-MLR system revealed that the immunosuppressive effect of trophoblast giant cells and trophoblast giant cell culture supernatants can be abolished with APS. Identical results were obtained with APS added to immunosuppressive doses of progesterone. CML between C57BL responder lymph node cells and mitomycin C-treated BALB/c stimulator spleen cells was also markedly suppressed when trophoblast of EPC origin was added. A similar suppression of cytotoxic T-cell induction was seen when progesterone was added to the system. The immunosuppressive action of trophoblast as detected in vitro is likely to play an important role in the maintainance of pregnancy by protecting the semiallogeneic conceptus against immune aggression by the maternal immune system.     

Differentiation of blastocysts derived from in vitro-fertilized rhesus monkey ova

Six in vitro-fertilized ova were cultured for 10 to 13 days in vitro. All six had formed blastocysts with cavities, five had hatched from the zona pellucida, and one had attached to the substrate. After fixation and preparation for examination by light and electron microscopy, it was determined that all but the youngest blastocysts had developed substantial amounts of syncytial trophoblast, which morphologically resembled the syncytial trophoblast present in the first 2 days of implantation in vivo. One of the smaller blastocysts had developed syncytial trophoblast but had not hatched from the zona. All of the blastocysts showed indications of loss or inadequate development of the inner cell mass constituents, indicating that the culture conditions were suboptimal for these constituents. Apparent C-type virus particles were abundant, budding from the basal surface of the syncytial trophoblast. Because the type of trophoblast formed was that normally associated with epithelial invasion and formation of the trophoblast plate, it is suggested that such blastocysts would be useful for in vitro implantation studies as well as studies of formation of syncytial trophoblast. However, other methods should be developed for study of postimplantation embryo development. It is also noted that the inadequate differentiation of the epiblast and endoderm would not have been apparent without microscopic examination.     

Recombinant entactin promotes mouse primary trophoblast cell adhesion and migration through the Arg-Gly-Asp (RGD) recognition sequence [published erratum appears in J Cell Biol 1993 Jul;122(1):279]

In vitro culture of mouse blastocysts during the period coinciding with implantation has revealed that primary trophoblast cells can adhere and migrate in serum-free medium when provided with certain extracellular matrix components, including fibronectin and laminin. Tightly associated with laminin is the glycoprotein, entactin, that may play an important role in basement membrane assembly and cell attachment. Mouse blastocysts were studied using this in vitro model to determine whether entactin was capable of mediating trophoblast invasive activity. Although entactin has never been shown to promote cell migration, we report here that recombinant entactin supported blastocyst outgrowth in a dose-dependent manner, with a maximal effect at 20-50 micrograms/ml. The ability of trophoblast cells to adhere and migrate on entactin was specifically inhibited by anti-entactin antibody, but not by antibodies raised against laminin. The synthetic peptide, Gly-Arg-Gly-Asp-Ser-Pro, that contains the Arg-Gly-Asp (RGD) integrin recognition site, reversibly inhibited entactin-mediated blastocyst outgrowth in a dose- dependent manner, but had no effect on laminin-mediated outgrowth. The synthetic peptide, Gly-Phe-Arg-Gly-Asp-Gly-Gln, that comprises the actual RGD-containing sequence within entactin, promoted trophoblast outgrowth when immobilized on the substratum. Furthermore, a mutated recombinant entactin, altered to contain a Glu in place of Asp at the RGD site, provided no trophoblast cell adhesive activity. We conclude that entactin promotes trophoblast outgrowth through a mechanism mediated by the RGD recognition site, and that it may play an important role during invasion of the endometrial basement membrane at implantation.     

Fibronectin and laminin promote in vitro attachment and outgrowth of mouse blastocysts

The process of mammalian implantation has been investigated using an in vitro model system wherein the trophoblast cells of mouse blastocysts attach to and outgrowth on tissue culture plates containing a complex medium. We now report that two extracellular matrix glycoproteins, fibronectin and laminin, when individually precoated on tissue culture plates promoted in vitro attachment and outgrowth of mouse blastocysts in serum-free medium. The kinetics of attachment and outgrowth processes in the presence of either of these two proteins were identical to that observed in complex, serum-containing medium. In contrast, plates containing a collagen matrix or pretreated with a variety of other serum proteins or various lectins failed to support in vitro attachment and outgrowth of blastocysts. Because all components of the culture medium are defined and both fibronectin and laminin are known components of the basement membrane of the endometrium, this in vitro system offers considerable advantages over the serum supplemented system to study in vitro implantation.     

Carbohydrate changes in pre- and peri-implantation mouse embryos as detected by a monoclonal antibody

We have examined the tissue and embryonic distribution of an antigen on a large polysaccharide that is recognized by a monoclonal antibody, IIC3, prepared against F9 teratocarcinoma cells. By immunofluorescence the antigen is first detected on compacted morulae and early blastocysts. It is strongly expressed on the primary endoderm and trophoblast of expanded blastocysts, but then disappears from the trophoblast of attached blastocysts in vitro. The binding of the antibody is completely inhibited by D-galactose and N-acetylgalactosamine. Fluoresceinated lectins were used to study further the changes in cell surface carbohydrates on trophoblast during implantation. Ricinus I, specific for terminal galactose, binds to preimplantation stages but does not bind to the trophoblast of the attached blastocyst. On the other hand, wheat germ agglutinin, specific for N-acetylglucosamine and sialic acid, binds to all preimplantation embryos and also to attached blastocysts (embryo proper and trophoblast). Neuraminidase treatment of blastocyst outgrowths enhances binding of both IIC3 and Ricinus I to the trophoblast; conversely, the binding of wheat germ agglutinin is decreased by this treatment. The results obtained in this study show changes of cell surface carbohydrates during early mouse development and suggest that sialic acid may be masking molecules on the surface of the trophoblast at the time of implantation.     

An ultrastructural study of in-vitro interaction of guinea-pig and mouse blastocysts with extracellular matrices.

Guinea-pig (intrusive) and mouse (displacement) blastocysts display different cellular mechanisms of implantation. Blastocysts were placed in CMRL-1066 supplemented with either 10 or 20% fetal calf serum, 0.1M L-glutamine and antibiotics and then transferred to dishes previously coated with either Matrigel or type I collagen. After culture for 48 or 72 h, the dishes were processed for transmission electron microscopy. Blastocysts had attached to both extracellular matrices by 48 h. Matrigel elicited minimal trophoblast cell activity. Trophoblast cell projections were oriented parallel to the Matrigel and displayed little invasive activity, but trophoblast cells displayed active interaction with type I collagen. By 72 h, trophoblast cells exhibited slender, anastomosing projections which extended into the collagen matrix. Bundles of microfilaments running parallel with the long axis of the projections were observed. The morphology of type I collagen was altered in the immediate vicinity of the trophoblast projections. The projections interdigitated and desmosomes developed between processes. Projections appeared to meet, fuse and entrap matrix. These results suggest that trophoblast cells do not significantly interact with Matrigel, but penetrate into type I collagen.     

Trophectoderm surface expression of the cell adhesion molecule cell-CAM 105 on rat blastocysts

A variety of cellular interactions is involved in the process of implantation of the mammalian embryo into the uterine tissue. Recent discoveries have demonstrated that intercellular recognition and adhesive events are governed by a class of cell surface molecules known as cell adhesion molecules (CAMs). In the present report, we have investigated the occurrence of the well-characterized cell adhesion molecule cell-CAM 105 on the surface of rat pre- and peri-implantation embryos of various stages. This was carried out by indirect immunofluorescence microscopy employing affinity-purified rabbit antibodies against cell-CAM 105. The embryonal stages investigated comprised morulae, normal day-4 blastocysts, and delayed and adhesive blastocysts obtained by using the method of experimentally delayed implantation. Cell-CAM 105 was absent in the early-morula stage, but in normal day-4 blastocysts and delayed blastocysts a specific staining for cell-CAM 105 was seen on the entire surface. However, adhesive-stage blastocysts exhibited a marked polarity with staining of the polar trophoblast cells. Scanning electron microscopy of adhesive-stage blastocysts revealed that the stronger staining of the polar region was not due to a greater number of microvilli on the polar trophoblast cells. Thus, it seems as if cell-CAM 105 is lost or masked from the surface of the mural trophoblast cells of adhesive-stage rat blastocysts. Since the mural trophoblast cells are the first to adhere to the uterine luminal epithelium during the onset of implantation and subsequently invade the uterine stroma, we suggest that the apparent downregulation of cell-CAM 105 in the mural trophoblast cells might be linked to the acquisition of trophoblast invasiveness.     

Development of amino acid transport system B0,+ in mouse blastocysts

The capacity of preimplantation mouse blastocysts to express the novel amino acid transport activity provisionally designated system B0,+ increased approximately 3-fold 1 day after administration of estrogen to their progesterone-primed, ovariectomized mothers. Nevertheless, blastocysts obtained 22-25 h after estrogen administration (implanting blastocysts) had to be incubated in vitro for about 20 min before they fully expressed their B0,+ activity. No similar increase in B0,+ activity was observed upon incubation of blastocysts obtained before estrogen administration (diapausing blastocysts). Rapid metabolic changes can be induced in the uterus by massaging it with a blunt instrument while it is receptive to implantation, and this treatment was found to increase the apparent B0,+ activity in implanting but not diapausing blastocysts. In contrast, the activity of an incompletely characterized, Na+-independent system, which accepts L-lysine as a substrate, decreased more than 2-fold when implanting blastocysts were incubated in vitro. No change in Na+-independent lysine uptake was detected during incubation of diapausing blastocysts. It is suggested that both uteri and blastocysts develop the capacity to change rapidly some of their metabolic processes near the time of implantation, and one of the processes which may be subject to rapid change in blastocysts is amino acid transport. These developmental events appear to coincide with and could be required for the decidual cell response and implantation of blastocysts in the uterus.     

Long Term Culture of Cells Derived from Mouse Blastocysts

The development of mouse blastocysts in primary culture has been followed for up to two months. The trophectoderm layer of the blastocyst gives rise to a monolayer of trophoblast cells; cells resembling both ectoplacental cone cells and primary giant cells are observed. The former can transform to giant cells, presumably secondary trophoblast, after several days in culture. Giant trophoblast cells are evident in the culture for much longer than the normal gestation period. Under the culture conditions described, the proportion of blastocysts showing substantial inner cell mass (ICM) proliferation in vitro is higher than that noted in previous studies. The ICM clumps develop into either egg cylinder-like structures, or, more commonly, into spherical, fluid-filled vesicles. The vesicles, which resemble yolk sac morphologically and biochemically [10, 11], continue to enlarge in size during several weeks of culture. The vesicles are attached to the underlying trophoblast monolayers by a stalk. Cells appear to migrate from this stalk out along the culture dish. The result after two to four weeks of culture is the appearance of a mixed monolayer containing a variety of different cell types. Secondary cultures of blastocyst cells have been continuously maintained in vitro for more than one year. Four lines of cells, all developing from the same pool of blastocysts, have been monitored for morphological, growth and biochemical properties, as well as chromosome number. Each line contained two or more morphologically distinct cell types, clearly indicated by cloning studies after eight months of culture. Doubling times and saturation densities among the four lines differed, as did biochemical properties. Although none of the cell lines resembled trophoblast biochemically after 7.5 months in culture, one line, MB4, possessed a number of biochemical properties in common with midgestation yolk sac. After a further five months of culture, some enzymes in the four lines were relatively unchanged; in other cases, notably with alkaline phosphatase, a sharp drop in enzyme activity was observed. One cell line, MB2, and specifically one of the cell types in this line, produced a yellow-orange pigment with a spectrum resembling that of a heme protein. After 7.5 months of culture, two of the four lines, MB21 and MB31, contained large numbers of cells with a diploid number of chromosomes. However, by 12.5 months in culture, the large majority of metaphases in all four cell lines possessed a hypotetraploid chromosome number. In a number of studies carried out to date, none of the cell lines generated tumors when injected into syngeneic hosts.     

Mouse blastocyst surface expression of galactose-containing epitopes coinciding with trophoblast differentiation

A galactose-containing cell surface epitope of mouse blastocysts was identified and partially characterized by means of immuno- and lectincytochemistry, using a mouse IgM anti-blastocyst monoclonal antibody (mAb N63) and four different galactose-binding lectins (BSL-1, DBA, PNA and SBA) as molecular probes. The mAb was produced by syngeneic intrasplenic immunization with adhesive mouse blastocysts, obtained 18 h after estrogen reactivation from facultative delay of implantation. Labelling of different mouse embryonic stages collected by uterine flushings revealed that the labelling of the epitope by monoclonal antibodies was restricted to the blastocyst stage. A peak labelling intensity was observed on late blastocysts. When examining blastocyst outgrowths, both trophoblast and embryoblast were weakly stained by mAb N63. Direct antigen characterization performed on blastocysts indicated that the mAb N63 recognized a galactose-containing glycolipid antigen. Immunochemistry of cryosectioned, unfixed mouse tissues including ovary, testis, uterus in delay and at implantation, Day 12 and term placenta, liver, kidney, brain, intestine, heart, striated muscle, and skin was negative. In addition, labelling of rat and hamster blastocysts was negative. In vitro experiments demonstrated that the galactose-containing blastocyst surface epitope was not involved in blastocyst attachment to plastic culture dishes. The appearance of the epitope at the embryonic surface in vivo coincides with the time of trophoblast differentiation and implantation in the mouse.     

Increased survival of preimplantation mouse embryos in medium with recombinant cytokine LIF

We studied the effects of cytokine LIF on in vitro development of 2-cell mouse embryos to the late blastocyst stage. LIF at 10 ng/ml enhanced the blastocyst formation and hatching from zona pellucida. When blastocysts were cultivated in a medium with LIF for a longer time, the trophoblast adhesive properties and proliferative activity were enhanced. In the presence of this cytokine, the trophoblast cells were attached to the substrate surface and fulfill the function of a sublayer for growth of the inner cell mass colonies with a high activity of endogenous alkaline phosphatase. Expression of LIF was detected in the oviduct and uterus epithelial tissues from day 1 until day 4 of pregnancy, thus suggesting its involvement in early development. According to the data of cultivation, cytokine LIF enhanced the adhesive properties and functional activity of the trophoblast cells, which is essential for implantation of blastocysts in the uterus.     

In vitro canine oocyte nuclear maturation in homologous oviductal cell co-culture with hormone-supplemented media

The aim of the present research was to verify the influence of oviductal cell co-culture previously supplemented with steroids (estrogen, progesterone, or both) on IVM rates for oocytes from anestrous bitches that were cultured in vitro for 48, 72 and 96 h. Oocytes harvested from anestrous bitches were selected and allocated into four groups: Group 1 (co-culture in oviductal epithelial cells without hormonal supplementation-control); Group 2 (estrogen supplementation); Group 3 (progesterone supplementation); Group 4 (estrogen+progesterone supplementation). The oviductal epithelial cell culture was established 72 h prior to oocyte co-culture. After periods of 48, 72 and 96 h, the degree of oocyte nuclear maturation was assessed. Co-culture in oviductal epithelial cells with estrogen was not as beneficial for canine IVM as supplementation with progesterone and estrogen, or progesterone supplementation alone. Therefore, it was feasible to use co-culture with oviductal epithelial cells obtained from anestrous bitches for IVM (monolayer culture with oviduct cells previously supplemented with progesterone). Final stages of oocyte maturation were achieved at 72 and 96 h of culture; therefore, the duration of maturation for oocytes obtained from bitches in different stages of the estrous cycle should be taken into account.     

Expression and function of cell surface extracellular matrix receptors in mouse blastocyst attachment and outgrowth

Mouse-hatched blastocysts cultured in vitro will attach and form outgrowths of trophoblast cells on appropriate substrates, providing a model for implantation. Immediately after hatching, the surfaces of blastocysts are quiescent and are not adhesive. Over the period 24-36 h post-hatching, blastocysts cultured in serum-free medium become adhesive and attach and spread on the extracellular matrix components fibronectin, laminin, and collagen type IV in a ligand specific manner. Attachment and trophoblast outgrowth on these substrates can be inhibited by addition to the culture medium of an antibody, anti-ECMr (anti-extracellular matrix receptor), that recognizes a group of 140-kD glycoproteins similar to those of the 140-kD extracellular matrix receptor complex (integrin) recognized in avian cells by CSAT and JG22 monoclonal antibodies. Addition to the culture medium of a synthetic peptide containing the Arg-Gly-Asp tripeptide cell recognition sequence of fibronectin inhibits trophoblast outgrowth on both laminin and fibronectin. However, the presence of the peptide does not affect attachment of the blastocysts to either ligand. Immunoprecipitation of 125I surface-labeled embryos using anti-ECMr reveals that antigens recognized by this antibody are exposed on the surfaces of embryos at a time when they are spreading on the substrate, but are not detectable immediately after hatching. Immunofluorescence experiments show that both the ECMr antigens and the cytoskeletal proteins vinculin and talin are enriched on the cell processes and ventral surfaces of trophectoderm cells in embryo outgrowths, in patterns similar to those seen in fibroblasts, and consistent with their role in adhesion of the trophoblast cells to the substratum.     

Uterine RGS2 expression is regulated by exogenous estrogen and progesterone in ovariectomized mice,and downregulation of RGS2 expression in artificial decidualized ESCs inhibits trophoblast spreading in vitro

Embryo implantation is a complicated event that relies on two critical factors: the competent blastocyst and the receptive uterus. Successful implantation results from tight coordination of these two factors. The maternal hormone environment of the uterus and molecular cross‐talk between the embryo and uterine tissue play pivotal roles in implantation. Here we showed that regulator of G‐protein signaling 2 (RGS2), a member of ubiquitous family of proteins that regulate G‐protein activation, plays an important role in embryo implantation by interfering in the cross‐talk between the embryo and uterine tissue. RGS2 expression increased during the implantation process, and was higher in the implant site than at the nonimplantation site. Meanwhile, ovariectomized (OVX) mice exhibited higher expression of RGS2 in the uterus. Exogenous 17β‐estradiol and progesterone in OVX mice downregulated the expression of RGS2. Treatment with exogenous 17β‐estradiol alone caused uterine RGS2 messenger RNA levels of OVX mice to return to those of normal female mice; when these mice were treated with progesterone or 17β‐estradiol plus progesterone, RGS2 levels rose. Downregulation of Rgs2 by small interfering RNA in an in vitro coculture system of decidualized endometrial stromal cells and blastocysts inhibited blastocyst outgrowth by restricting trophoblast spreading, suggesting a mechanism by which RGS2 regulates embryo implantation.     

Scanning electron microscopy of the surface of normal and implantation-delayed mouse blastocysts during development in vitro

Mouse blastocysts undergo developmental steps in culture analogous to those occurring during implantation in utero. We examined cultured blastocysts by scanning electron microscopy (SEM) as they passed through these stages. From the time of hatching to the acquisition of adhesiveness, most blastocysts were exhanded, with flattened cells possessing relatively small numbers of microvilli, centrally raised areas (presumably reflecting the location of the nuclei) and intercellular ridges often possessing microvilli. At, or shortly before, the trophoblast outgrowth stage, blastocysts appeared to contract; the cells bulged noticeably, microvilli covered the entire surface of most cells and intercellular ridges were no longer observable. Blastocysts removed from uteri on the seventh day of ovariectomy delay possessed a variety of morphologies and shapes. The blastocoel was frequently collapsed and cell outlines were difficult to discern. These blastocysts were initially adhesive in vitro, but subsequently disengaged from the substratum before becoming permanently adherent several hours later. During the initial phase of adhesiveness, blastocysts were elongated and had prominent intercellular ridges, particularly in the equatorial region. Detached blastocysts contained bulging cells with contours which obscured the intercellular ridges. Surface ultrastructure during subsequent phases resembled non-delayed blastocysts during attachment and outgrowth. On the basis of our studies, we propose that intercellular ridges play some role in blastocyst adhesiveness. However, we must conclude that there are other factors involved in the acquisition of adhesiveness by the blastocyst which are at least equally important but of a nature too subtle to be identified by our SEM analyses. Insofar as delayed blastocysts are concerned, we find that, within limits, the surface alterations that take place when blastocysts are activated in culture mirror those observed following reversal of delay in vivo by administration of hormones. Since delayed blastocysts placed in saline also undergo morphological changes resembling those seen at the onset of activation in utero, we suggest that reversal of implantation delay requires initially neither direct contact with steroid or macromolecular inducers nor an exogenous supply of metabolites.     

层粘连蛋白及其肽段对小鼠胚泡粘附和扩展的作用

作为细胞外基质的主要成分之一的层粘连蛋白（ＬＮ）,对小鼠胚泡的粘附、扩展有显著促进作用。ＬＮ分子上的一些活性位点对胚泡的粘附和扩展也具有一定的作用,含ＲＧＤ位点序列的合成肽段ＲＧＤＳ对胚泡的粘附有促进作用;含ＹＩＧＳＲ位点序列的合成肽段ｃＹＩＧＳＲ对胚泡的粘附和扩展均有促进作用;且ＲＧＤＳ和ｃＹＩＧＳＲ可以竞争性抑制ＬＮ对胚泡粘附和扩展的促进作用。以上结果表明ＬＮ对胚泡的作用是通过胚泡上不同的ＬＮ     

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

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 more than 25,000 to more than 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 NH(4)OH 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 degredation 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, 17β-estradiol, leupeptin, EDTA, colchicine, NH(4)Cl, or ε-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.0     

The effects of soluble products of activated lymphocytes and macrophages on blastocyst implantation events in vitro

The purpose of this study was to investigate the effects of soluble products of activated lymphocytes and macrophages on mouse blastocyst attachment and trophoblast outgrowth in vitro. Hatched blastocysts were incubated with medium alone, supernatant fluids from mixed lymphocyte cultures (MLC), and with individual human and murine lymphokines and monokines in fibronectin-coated wells. Cultures were assessed at 24, 48, and 72 h for blastocyst attachment and trophoblast outgrowth. Both human and murine MLC supernatant fluids significantly enhanced trophoblast outgrowth in vitro. The cytokine, interleukin-1 beta (Il-1 beta), at a concentration of 10(3) U/ml, inhibited blastocyst attachment but significantly enhanced trophoblast outgrowth of attached blastocysts. Granulocyte, macrophage-colony-stimulating factor (GM-CSF) at a concentration of 250 U/ml significantly inhibited blastocyst attachment, while gamma interferon (gamma-IFN) at a concentration of 2.5 x 10(3) U/ml significantly inhibited trophoblast outgrowth and caused degenerative morphological changes in these cells. The results of this study indicate that products of activated immune cells may either facilitate or impede implantation events depending on the types of predominant cytokines present, their concentration(s), and the timing of their secretion relative to embryonic development.     

Matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-3 are key regulators of extracellular matrix degradation by mouse embryos

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.